RNA preparations comprising purified modified RNA for reprogramming cells

ABSTRACT

The present invention provides compositions and methods for reprogramming somatic cells using purified RNA preparations comprising single-strand mRNA encoding an iPS cell induction factor. The purified RNA preparations are preferably substantially free of RNA contaminant molecules that: i) would activate an immune response in the somatic cells, ii) would decrease expression of the single-stranded mRNA in the somatic cells, and/or iii) active RNA sensors in the somatic cells. In certain embodiments, the purified RNA preparations are substantially free of partial mRNAs, double-stranded RNAs, un-capped RNA molecules, and/or single-stranded run-on mRNAs.

The present application is a continuation of U.S. patent applicationSer. No. 15/160,062, filed May 20, 2016, now U.S. Pat. No. 10,006,007,which is a continuation of U.S. patent application Ser. No. 14/801,075,filed Jul. 16, 2015, now U.S. Pat. No. 9,371,511, which is acontinuation of U.S. patent application Ser. No. 14/644,380, filed Mar.11, 2015, now U.S. Pat. No. 9,163,213, which is a continuation of U.S.patent application Ser. No. 12/962,468, filed Dec. 7, 2010, now U.S.Pat. No. 9,012,219, which claims priority to U.S. ProvisionalApplication Ser. No. 61/267,312 filed Dec. 7, 2009, each of which isherein incorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to compositions and methods for changingor reprogramming the state of differentiation of eukaryotic cells,including human or other animal cells, by contacting the cells withpurified RNA preparations comprising or consisting of one or moredifferent single-strand mRNA molecules that each encode a reprogrammingfactor (e.g., an iPS cell induction factor). The purifiedsingle-stranded mRNA molecules preferably comprise at least one modifiednucleoside (e.g., selected from the group consisting of a pseudouridine(abbreviated by the Greek letter “psi” or “ψ”), 5-methylcytosine (m⁵C),5-methyluridine (m⁵U), 2′-O-methyluridine (Um or m^(2′-O)U),2-thiouridine (s²U), and N⁶-methyladenosine (m⁶A)) in place of at leasta portion of the corresponding unmodified canonical nucleoside (e.g., inplace of substantially all of the corresponding unmodified A, C, G, or Tcanonical nucleoside). In addition, the single-stranded mRNA moleculesare preferably purified to be substantially free of RNA contaminantmolecules that would activate an unintended response, decreaseexpression of the single-stranded mRNA, and/or activate RNA sensors inthe cells. In certain embodiments, the purified RNA preparations aresubstantially free of RNA contaminant molecules that are: shorter orlonger than the full-length single-stranded mRNA molecules,double-stranded, and/or uncapped RNA.

BACKGROUND

In 2006, it was reported (Takahashi and Yamanaka 2006) that theintroduction of genes encoding four protein factors (OCT4 (Octamer-4;POU class 5 homeobox 1), SOX2 (SRY (sex determining region Y)-box 2),KLF4 (Krueppel-like factor 4), and c-MYC) into differentiated mousesomatic cells induced those cells to become pluripotent stem cells,(referred to herein as “induced pluripotent stem cells,” “iPS cells,” or“iPSCs”). Following this original report, pluripotent stem cells werealso induced by transforming human somatic cells with genes encoding thesimilar human protein factors (OCT4, SOX2, KLF4, and c-MYC) (Takahashiet al. 2007), or by transforming human somatic cells with genes encodinghuman OCT4 and SOX2 factors plus genes encoding two other human factors,NANOG and LIN28 (Lin-28 homolog A) (Yu et al. 2007). All of thesemethods used retroviruses or lentiviruses to integrate genes encodingthe reprogramming factors into the genomes of the transformed cells andthe somatic cells were reprogrammed into iPS cells only over a longperiod of time (e.g., in excess of a week).

The generation iPS cells from differentiated somatic cells offers greatpromise as a possible means for treating diseases through celltransplantation. The possibility to generate iPS cells from somaticcells from individual patients also may enable development ofpatient-specific therapies with less risk due to immune rejection. Stillfurther, generation of iPS cells from disease-specific somatic cellsoffers promise as a means to study and develop drugs to treat specificdisease states (Ebert et al. 2009, Lee et al. 2009, Maehr et al. 2009).

Viral delivery of genes encoding protein reprogramming factors (or “iPSCfactors”) provides a highly efficient way to make iPS cells from somaticcells, but the integration of exogenous DNA into the genome, whetherrandom or non-random, creates unpredictable outcomes and can ultimatelylead to cancer (Nakagawa et al. 2008). New reports show that iPS cellscan be created (at lower efficiency) by using other methods that do notrequire genome integration. For example, repeated transfections ofexpression plasmids containing genes for OCT4, SOX2, KLF4 and c-MYC intomouse embryonic fibroblasts to generate iPS cells was demonstrated(Okita et al. 2008). Induced pluripotent stem cells were also generatedfrom human somatic cells by introduction of a plasmid that expressedgenes encoding human OCT4, SOX2, c-MYC, KLF4, NANOG and LIN28 (Yu et al.2009). Other successful approaches for generating iPS cells includetreating somatic cells with: recombinant protein reprogramming factors(Zhou et al. 2009); non-integrating adenoviruses (Stadtfeld et al.2008); or piggyBac transposons (Woltjen et al. 2009) to deliverreprogramming factors. Presently, the generation of iPS cells usingthese non-viral delivery techniques to deliver reprogramming factors isextremely inefficient. Future methods for generating iPS cells forpotential clinical applications will need to increase the speed andefficiency of iPS cell formation while maintaining genome integrity.

SUMMARY OF THE INVENTION

The present invention provides compositions and methods forreprogramming the state of differentiation of eukaryotic cells,including human or other animal cells, by contacting the cells withpurified RNA preparations comprising or consisting of one or moredifferent single-strand mRNA molecules that each encode a reprogrammingfactor (e.g., an iPS cell induction factor). The purifiedsingle-stranded mRNA molecules preferably comprise at least one modifiednucleoside (e.g., selected from the group consisting of a pseudouridine(ψ), 5-methylcytosine (m⁵C), 5-methyluridine (m⁵U), 2′-O-methyluridine(Um or m^(2′-O) U), 2-thiouridine (s²U), and N⁶-methyladenosine (m⁶A))in place of at least a portion (e.g., including substantially all) ofthe corresponding unmodified canonical nucleoside of the correspondingunmodified A, C, G, or T canonical nucleoside. In addition, thesingle-stranded mRNA molecules are preferably purified to besubstantially free of RNA contaminant molecules that would activate anunintended response, decrease expression of the single-stranded mRNA,and/or activate RNA sensors (e.g., double-stranded RNA-dependentenzymes) in the cells. In certain embodiments, the purified RNApreparations are substantially free of RNA contaminant molecules thatare: shorter or longer than the full-length single-stranded mRNAmolecules, double-stranded, and/or uncapped RNA. In some preferredembodiments, the invention provides compositions and methods forreprogramming differentiated eukaryotic cells, including human or otheranimal somatic cells, by contacting the cells with purified RNApreparations comprising or consisting of one or more differentsingle-strand mRNA molecules that each encode an iPS cell inductionfactor.

In some embodiments, the present invention provides methods for changingthe state of differentiation of a somatic cell comprising: introducingan mRNA encoding an iPS cell induction factor into a somatic cell togenerate a reprogrammed dedifferentiated cell, wherein the mRNAcomprises at least one 5-methylcytidine (or other modified baseddescribed herein).

In certain embodiments, the present invention provides methods forreprogramming a cell that exhibits a first differentiated state orphenotype to a cell that exhibits a second differentiated state orphenotype comprising: introducing into the cell that exhibits a firstdifferentiated state a purified RNA preparation comprising modified mRNAmolecules that encode at least one reprogramming factor and culturingthe cell under conditions wherein the cell exhibits a seconddifferentiated state. In certain embodiments, the modified mRNAmolecules contain at least one modified nucleoside selected from thegroup consisting of psuedouridine or 5-methylcytidine. In certainembodiments, the cell is from a human or animal. In further embodiments,the purified RNA preparation: i) comprises first single-stranded mRNAsencoding a first iPS cell induction factor, wherein substantially all ofthe first single-stranded complete mRNAs comprise at least onepseudouridine residue and/or at least one 5-methylcytidine residue, andii) is substantially free of RNA contaminant molecules which are able toactivate RNA sensors in said somatic cell. In certain embodiments, theRNA contaminant molecules are selected from the group consisting of:partial mRNAs encoding only a portion of said iPS cell induction factor,RNA molecules that are smaller than the full-length mRNA, RNA moleculesthat are larger than the full-length mRNA, double-stranded mRNAmolecules, and un-capped mRNA molecules.

In some embodiments, the present invention provides methods forreprogramming a somatic cell (e.g., dedifferentiating ortransdifferentiating) comprising: contacting a somatic cell with apurified RNA preparation to generate a reprogrammed cell, wherein thepurified RNA preparation: i) comprises first single-stranded mRNAsencoding a first iPS cell induction factor, wherein substantially all ofthe first single-stranded complete mRNAs comprise at least onepseudouridine residue and/or at least one 5-methylcytidine residue, andii) is substantially free of contaminant molecules (e.g., RNAcontaminant molecules) which are able to activate RNA sensors in thesomatic cell. In particular embodiments, the RNA contaminant moleculescomprise: partial mRNAs encoding only a portion of the iPS cellinduction factor, single-stranded run-on mRNAs encoding the iPS cellinduction factor and encoding at least an additional portion of the iPScell induction factor, double-stranded mRNA molecules, and un-cappedmRNA molecules. In certain embodiments, the first single-stranded mRNAsdo not also encoding an additional portion of the first iPS cellinduction factor.

In some embodiments, the reprogrammed cell is a dedifferentiated cell(e.g., stem cell or stem cell-like cell). In other embodiments, thereprogrammed cell is a transdifferentiated cells (e.g., a skin cells isreprogrammed into a neuronal cell, or other type of change). In furtherembodiments, the first single-stranded mRNAs encode the complete firstiPS induction factor (e.g, the mRNA encodes the entire coding sequencefor a particular iPS induction factor). In other embodiments, thecontacting further comprises contacting the somatic cell with a growthfactor and/or cytokine (e.g, after a period of time). In furtherembodiments, the contact further comprises contacting the somatic cellwith an immune response inhibitor.

In certain embodiments, all or nearly all of the uridine nucleosides inthe first single-stranded mRNA are replaced by pseudouridinenucleosides. In other embodiments, all or nearly all of the cytidinenucleosides in the first single-stranded mRNA are replaced by5-methylcytidine nucleosides or another base recited herein.

In particular embodiments, the present invention provides methods forgenerating a reprogrammed cell comprising: contacting a somatic cellwith a purified RNA preparation to generate a reprogrammed cell that isable to survive in culture for at least 10 days (e.g., at least 10 days. . . at least 13 days . . . at least 16 days . . . at least 20 days . .. at least 40 days . . . or is able to form a cell-line), wherein thepurified RNA preparation comprises first single-stranded mRNAs encodingan iPS cell induction factor, and wherein a majority of the firstsingle-stranded mRNAs comprise at least one pseudouridine residue and/orat least one 5-methylcytidine residue.

In certain embodiments, the purified RNA preparation is free of anamount of RNA contaminant molecules that would activate an immuneresponse in the somatic cell sufficient to prevent the reprogrammed cellfrom surviving at least 10 days in culture (e.g., at least 10 days . . .at least 15 days . . . at least 20 days . . . at least 40 days, orlonger). In other embodiments, the RNA contaminant molecules include:partial mRNAs encoding only a portion of the iPS cell induction factor,single-stranded run-on mRNAs fully encoding the iPS cell inductionfactor and encoding at least an additional portion of the iPS cellinduction factor, double-stranded mRNA molecules, un-capped mRNAmolecules, and mixtures thereof. In certain embodiments, thereprogrammed cell that is generated is able to form a reprogrammedcell-line. In other embodiments, the purified RNA preparation is free ofan amount of RNA contaminant molecules that would activate an immuneresponse in the somatic cell sufficient to prevent generation of thereprogrammed cell-line.

In particular embodiments, the RNA contaminant molecules are selectedfrom the group consisting of: partial mRNAs encoding only a portion ofthe iPS cell induction factor, single-stranded run-on mRNAs encoding theiPS cell induction factor and encoding at least an additional portion ofthe iPS cell induction factor, double-stranded mRNA molecules, un-cappedmRNA molecules, and mixtures thereof.

In some embodiments, the present invention provides methods forgenerating a reprogrammed cell-line comprising: a) contacting a somaticcell with a purified RNA preparation to generate a reprogrammed cell,wherein the purified RNA preparation comprises mRNAs encoding an iPScell induction factor, and wherein a majority of the mRNAs comprise atleast one pseudouridine residue and/or at least one 5-methylcytidineresidue, and b) culturing the dedifferentiated cell to generate areprogrammed cell-line. In other embodiments, the purified RNApreparation is free of an amount of contaminant molecules that wouldactivate an immune response in the somatic cell sufficient to preventgeneration of the reprogrammed cell-line. In certain embodiments, theimmune response involves activation of RNA sensors in the somatic cell.

In some embodiments, the present invention provides methods forreprogramming a somatic cell comprising: contacting a somatic cell witha purified RNA preparation to generate a reprogrammed cell, wherein thepurified RNA preparation: i) comprises first single-stranded mRNAsencoding a first iPS cell induction factor, wherein substantially all ofthe first single-stranded mRNAs comprise at least one pseudouridineresidue and/or at least one 5-methylcytidine residue, and ii) issubstantially free of: a) partial mRNAs encoding only a portion of thefirst iPS cell induction factor, and b) double-stranded mRNA molecules.In further embodiments, the first single-stranded mRNA do not alsoencode an additional portion of the first iPS cell induction factor. Inparticular embodiments, the first single-stranded mRNA fully encode thefirst iPS cell induction factor. In other embodiments, the purified RNApreparation is also substantially free (or essentially free or virtuallyfree or free) of single-stranded run-on mRNAs encoding the first iPScell induction factor and encoding at least an additional portion of thefirst iPS cell induction factor. In other embodiments, the substantiallyall of the first single-stranded complete mRNAs are 5′ capped. In otherembodiments, the purified RNA preparation is also substantially free ofun-capped mRNA molecules. In some embodiments, substantially all of thefirst single-stranded mRNAs comprise at least one psuedouridine residue.In additional embodiments, substantially all of the firstsingle-stranded mRNAs comprise at least one 5-methylcytidine residue. Inother embodiments, substantially all of the first single-stranded mRNAscomprise at least one psuedouridine residue and at least one5-methycytidine residue.

In certain embodiments, the purified RNA preparation comprises atransfection reagent. In other embodiments, the purified RNA preparationis obtained by HPLC purification of an RNA sample that contains asubstantial amount of the partial mRNAs and the double-stranded mRNAs.In further embodiments, the purified RNA preparation is essentially freeof the partial mRNAs and the single-stranded run-on mRNAs. In someembodiments, the purified RNA preparation is essentially free orvirtually free or free of double-stranded mRNA molecules. In otherembodiments, the purified RNA preparation is essentially free orvirtually free or free of un-capped mRNA molecules. In some embodiments,substantially all of the first single-stranded mRNAs are polyadenylated.In other embodiments, the first single-stranded complete mRNAs arecapped with 7-methylguanosine.

In some embodiments, the first iPS cell induction factor is selectedfrom the group consisting of KLF4, LIN28, c-MYC, NANOG, OCT4, and SOX2.In other embodiments, the purified RNA preparation: i) further comprisessecond single-stranded mRNAs encoding a second iPS cell inductionfactor, wherein the second single-stranded mRNAs comprise at least onepseudouridine residue and/or at least one 5-methylcytidine residue, andii) is further substantially free of: a) partial mRNAs encoding only aportion of the second iPS cell induction factor, and b) double-strandedmRNAs. In other embodiments, the purified RNA preparation is furthersubstantially free of single-stranded run-on mRNAs encoding a second iPScell induction factor and encoding at least an additional portion of thesecond iPS cell induction factor. In some embodiments, the second iPScell induction factor is selected from the group consisting of KLF4,LIN28, c-MYC, NANOG, OCT4, and SOX2. In certain embodiments, the somaticcell is a fibroblast. In other embodiments, the reprogrammed cell is apluripotent stem cell. In other embodiments, the dedifferentiated cellexpresses NANOG and TRA-1-60. In some embodiments, the cell is in vitro.In further embodiments, the cell resides in culture. In particularembodiments, the cell resides in MEF-conditioned medium.

In some embodiments, the present invention provides compositionscomprising a purified RNA preparation, wherein the purified RNApreparation: i) comprises first single-stranded mRNAs encoding a firstiPS cell induction factor, wherein the first single-stranded mRNAscomprise at least one pseudouridine residue and/or at least one5-methylcytidine residue, and ii) is substantially free of RNAcontaminant molecules, which are able to activate RNA sensors in asomatic cell. In certain embodiments, the present invention providescompositions comprising a purified RNA preparation, wherein the purifiedRNA preparation: i) comprises first single-stranded mRNAs encoding afirst iPS cell induction factor, wherein the first single-strandedcomplete mRNAs comprise at least one pseudouridine residue and/or atleast one 5-methylcytidine residue, and ii) is substantially free of: a)partial mRNAs encoding only a portion of the first iPS cell inductionfactor, and b) double-stranded RNA.

In certain embodiments, the purified RNA preparation is alsosubstantially free of single-stranded run-on mRNAs encoding the firstiPS cell induction factor and encoding at least an additional portion ofthe first iPS cell induction factor. In some embodiments, the purifiedRNA preparation: i) further comprises second single-stranded mRNAsencoding a second iPS cell induction factor, wherein the secondsingle-stranded complete mRNAs comprise at least one pseudouridineresidue and/or at least one 5-methylcytidine residue, and ii) issubstantially free of: a) partial mRNAs encoding only a portion of thesecond iPS cell induction factor, and b) single-stranded run-on mRNAsencoding second first iPS cell induction factor and encoding at least anadditional portion of the second iPS cell induction factor.

In some embodiments, the present invention provides compositionscomprising an in vitro-synthesized mRNA encoding the MYC gene, whereinthe in vitro-synthesized mRNA comprises at least one pseudouridineresidue and/or at least one 5-methylcytidine residue. In certainembodiments, the compositions are substantially free of RNA contaminantmolecules which are able to activate RNA sensors in a somatic cell.

In particular embodiments, the present invention provides methods forinducing a mammalian cell to produce the MYC protein comprising:contacting a mammalian cell with an in vitro-synthesized mRNA encodingthe MYC gene, wherein the in vitro-synthesized mRNA comprises at leastone pseudouridine residue and/or at least one 5-methylcytidine residue,thereby inducing the mammalian cell to produce the MYC protein. In otherembodiments, the mammalian cell is a dendritic cell. In otherembodiments, the mammalian cell is an alveolar cell, an astrocyte, amicroglial cell, or a neuron.

In some embodiments, the present invention provides methods of treatinga subject comprising contacting a subject with the MYC protein producingmammalian cell described above and herein.

In additional embodiments, the present invention provides methods ofsynthesizing an in vitro-transcribed RNA molecule encoding the MYC genecomprising: combining an isolated RNA polymerase, a template nucleicacid sequence encoding the MYG gene, unmodified nucleotides, andpseudouridine or 5-methylcytidine modified nucleotides under conditionssuch that an in vitro-transcribed RNA molecule encoding the MYC gene isgenerated that comprises at least one pseudouridine or 5-methylcytidineresidue.

Experiments conducted during the development of embodiments of thepresent invention demonstrated that mRNA molecules can be administeredto cells and induce a dedifferentiation process to generatededifferentiated cells—including pluripotent stem cells. Thus, thepresent invention provides compositions and methods for generating iPScells. Surprisingly, the administration of mRNA can provide highlyefficient generation of iPS cells.

The present invention also provides RNA, oligoribonucleotide, andpolyribonucleotide molecules comprising pseudouridine or a modifiednucleoside, gene therapy vectors comprising same, methods ofsynthesizing same, and methods for gene replacement, gene therapy, genetranscription silencing, and the delivery of therapeutic proteins totissue in vivo, comprising the molecules. The present invention alsoprovides methods of reducing the immunogenicity of RNA,oligoribonucleotide, and polyribonucleotide molecules.

In some embodiments, the present invention provides methods fordedifferentiating a somatic cell comprising: introducing mRNA encodingone or more iPSC induction factors into a somatic cell to generate adedifferentiated cell.

In some embodiments, the present invention provides methods fordedifferentiating a somatic cell comprising: introducing mRNA encodingone or more iPSC induction factors into a somatic cell and maintainingthe cell under conditions wherein the cell is viable and the mRNA thatis introduced into the cell is translated in sufficient amount and forsufficient time to generate a dedifferentiated cell. In some preferredembodiments, the dedifferentiated cell is an induced pluripotent stemcell (iPSC).

In some embodiments, the present invention provides methods for changingthe state of differentiation (or differentiated state) of a eukaryoticcell comprising: introducing mRNA encoding one or more reprogrammingfactors into a cell and maintaining the cell under conditions whereinthe cell is viable and the mRNA that is introduced into the cell istranslated in sufficient amount and for sufficient time to generate acell that exhibits a changed state of differentiation compared to thecell into which the mRNA was introduced.

In some embodiments, the present invention provides methods for changingthe state of differentiation of a eukaryotic cell comprising:introducing mRNA encoding one or more reprogramming factors into a celland maintaining the cell under conditions wherein the cell is viable andthe mRNA that is introduced into the cell is translated in sufficientamount and for sufficient time to generate a cell that exhibits achanged state of differentiation compared to the cell into which themRNA was introduced. In some embodiments, the changed state ofdifferentiation is a dedifferentiated state of differentiation comparedto the cell into which the mRNA was introduced. For example, in someembodiments, the cell that exhibits the changed state of differentiationis a pluripotent stem cell that is dedifferentiated compared to asomatic cell into which the mRNA was introduced (e.g., a somatic cellthat is differentiated into a fibroblast, a cardiomyocyte, or anotherdifferentiated cell type). In some embodiments, the cell into which themRNA is introduced is a somatic cell of one lineage, phenotype, orfunction, and the cell that exhibits the changed state ofdifferentiation is a somatic cell that exhibits a lineage, phenotype, orfunction that is different than that of the cell into which the mRNA wasintroduced; thus, in these embodiments, the method results intransdifferentiation (Graf and Enver 2009).

The methods of the invention are not limited with respect to aparticular cell into which the mRNA is introduced. In some embodimentsof any of the above methods, the cell into which the mRNA is introducedis derived from any multi-cellular eukaryote. In some embodiments of anyof the above methods, the cell into which the mRNA is introduced isselected from among a human cell and another animal cell. Although thework presented herein was performed using cells of humans or otheranimals, the applicants further claim that the methods of the presentinvention comprising reprogramming human and animal cells by contactingthe cells with a purified RNA preparation that consists of one or morepurified single-stranded mRNA molecules, each of which encodes a proteinreprogramming factor (e.g., a transcription factor) also pertains toreprogramming of other eukaryotic cells (e.g., plant cells and a fungalcells). In some embodiments of any of the above methods, the cell intowhich the mRNA is introduced is a normal cell that is from an organismthat is free of a known disease. In some embodiments of any of the abovemethods, the cell into which the mRNA is introduced is a cell from anorganism that has a known disease. In some embodiments of any of theabove methods, the cell into which the mRNA is introduced is a cell thatis free of a known pathology. In some embodiments of any of the abovemethods, the cell into which the mRNA is introduced is a cell thatexhibits a disease state or a known pathology (e.g., a cancer cell, or apancreatic beta cell that exhibits metabolic properties characteristicof a diabetic cell).

The invention is not limited to the use of a specific cell type (e.g.,to a specific somatic cell type) in embodiments of the methodscomprising introducing mRNA encoding one or more iPSC cell inductionfactors in order to generate a dedifferentiated cell (e.g., an iPScell). Any cell that is subject to dedifferentiation using iPS cellinduction factors is contemplated. Such cells include, but are notlimited to, fibroblasts, keratinocytes, adipocytes, lymphocytes,T-cells, B-Cells, cells in mononuclear cord blood, buccal mucosa cells,hepatic cells, HeLa, MCF-7 or other cancer cells. In some embodiments,the cells reside in vitro (e.g., in culture) or in vivo. In someembodiments, when generated in culture, a cell-free conditioned medium(e.g., MEF-conditioned medium) is used. As demonstrated below, such amedium provided enhanced iPS cell generation. The invention is notlimited, however, to the culturing conditions used. Any culturingcondition or medium now known or later identified as useful for themethods of the invention (e.g., to generate iPS cells from somatic cellsand maintain said cells) is contemplated for use with the invention. Forexample, although not preferred, in some embodiments of the method, afeeder cell layer is used instead of conditioned medium for culturingthe cells that are treated using the method.

In some embodiments of any of these methods, the step of introducingmRNA comprises delivering the mRNA into the cell (e.g., a human or otheranimal somatic cell) with a transfection reagent (e.g., TRANSIT™ mRNAtransfection reagent, MirusBio, Madison, Wis.).

However, the invention is not limited by the nature of the transfectionmethod utilized. Indeed, any transfection process known, or identifiedin the future that is able to deliver mRNA molecules into cells in vitroor in vivo, is contemplated, including methods that deliver the mRNAinto cells in culture or in a life-supporting medium, whether said cellscomprise isolated cells or cells comprising a eukaryotic tissue ororgan, or methods that deliver the mRNA in vivo into cells in anorganism, such as a human, animal, plant or fungus. In some embodiments,the transfection reagent comprises a lipid (e.g., liposomes, micelles,etc.). In some embodiments, the transfection reagent comprises ananoparticle or nanotube. In some embodiments, the transfection reagentcomprises a cationic compound (e.g., polyethylene imine or PEI). In someembodiments, the transfection method uses an electric current to deliverthe mRNA into the cell (e.g., by electroporation). In some embodiments,the transfection method uses a bolistics method to deliver the mRNA intothe cell (e.g., a “gene gun” or biolistic particle delivery system.)

The data presented herein shows that, with respect to the mRNAintroduced into the cell, certain amounts of the mRNAs used in theEXAMPLES described herein resulted in higher efficiency and more rapidinduction of pluripotent stem cells from the particular somatic cellsused than other amounts of mRNA. However, the methods of the presentinvention are not limited to the use of a specific amount of mRNA tointroduce into the cell. For example, in some embodiments, a total ofthree doses, with each dose comprising 18 micrograms of each of sixdifferent mRNAs, each encoding a different human reprogramming factor,was used to introduce the mRNA into approximately 3×10⁵ human fibroblastcells in a 10-cm plate (e.g., delivered using a lipid-containingtransfection reagent), although in other embodiments, higher or loweramounts of the mRNAs were used to introduce into the cells.

The invention is not limited to a particular chemical form of the mRNAused, although certain forms of mRNA may produce more efficient results.However, in some preferred embodiments, the mRNA comprises at least onemodified nucleoside (e.g., selected from the group consisting of apseudouridine (ψ), 5-methylcytosine (m⁵C), 5-methyluridine (m⁵U),2′-O-methyluridine (Um or m^(2′-O)U), 2-thiouridine (s²U), andN⁶-methyladenosine (m⁶A)) in place of at least a portion of thecorresponding unmodified canonical nucleoside (e.g., in some preferredembodiments, at least one modified nucleoside in place of substantiallyall of the corresponding unmodified A, C, G, or T canonical nucleoside).In some embodiments, the mRNA is polyadenylated. In some preferredembodiments, the mRNA is prepared by polyadenylation of an invitro-transcribed (IVT) RNA, the method comprising contacting the IVTRNA using a poly(A) polymerase (e.g., yeast RNA polymerase or E. colipoly(A) polymerase). In some embodiments, the mRNA is polyadenylatedduring IVT by using a DNA template that encodes the poly(A) tail.Regardless of whether the RNA is polyadenylated using a poly(A)polymerase or during IVT of a DNA template, in some preferredembodiments, the mRNA comprises a poly-A tail (e.g., a poly-A tailhaving 50-200 nucleotides, e.g., preferably 100-200, 150-200nucleotides, or greater than 150 nucleotides), although in someembodiments, a longer or a shorter poly-A tail is used. In someembodiments, the mRNA used in the methods is capped. To maximizeefficiency of expression in the cells, it is preferred that the majorityof mRNA molecules contain a cap. In some preferred embodiments, the mRNAmolecules used in the methods are synthesized in vitro by incubatinguncapped primary RNA in the presence a capping enzyme system. In somepreferred embodiments, the primary RNA used in the capping enzymereaction is synthesized by in vitro transcription (IVT) of a DNAmolecule that encodes the RNA to be synthesized. The DNA that encodesthe RNA to be synthesized contains an RNA polymerase promoter, to which,an RNA polymerase binds and initiates transcription therefrom. It isalso known in the art that mRNA molecules often have regions ofdiffering sequence located before the translation start codon and afterthe translation stop codon that are not translated. These regions,termed the five prime untranslated region (5′ UTR) and three primeuntranslated region (3′ UTR), respectively, can affect mRNA stability,mRNA localization, and translational efficiency of the mRNA to whichthey are joined. Certain 5′ and 3′ UTRs, such as those for alpha andbeta globins are known to improve mRNA stability and expression ofmRNAs. Thus, in some preferred embodiments, the mRNAs that encodereprogramming factors (e.g., iPSC induction factors) exhibit a 5′ UTRand/or a 3′ UTR that results in greater mRNA stability and higherexpression of the mRNA in the cells (e.g., an alpha globin or a betaglobin 5′ UTR and/or 3′ UTR; e.g., a Xenopus or human alpha globin or abeta globin 5′ UTR and/or 3′ UTR, or, e.g., a tobacco etch virus (TEV)5′ UTR).

The IVT can be performed using any RNA polymerase as long as synthesisof the mRNA from the DNA template that encodes the RNA is specificallyand sufficiently initiated from a respective cognate RNA polymerasepromoter and full-length mRNA is obtained. In some preferredembodiments, the RNA polymerase is selected from among T7 RNApolymerase, SP6 RNA polymerase and T3 RNA polymerase. In some otherembodiments, capped RNA is synthesized co-transcriptionally by using adinucleotide cap analog in the IVT reaction (e.g., using an AMPLICAP™ T7Kit or a MESSAGEMAX™ T7 ARCA-CAPPED MESSAGE Transcription Kit; EPICENTREor CellScript, Madison, Wis., USA). If capping is performedco-transcriptionally, preferably the dinucleotide cap analog is ananti-reverse cap analog (ARCA). However, use of a separate IVT reaction,followed by capping with a capping enzyme system, which results inapproximately 100% of the RNA being capped, is preferred overco-transcriptional capping, which typically results in only about 80% ofthe RNA being capped. Thus, in some preferred embodiments, a highpercentage of the mRNA molecules used in a method of the presentinvention are capped (e.g., greater than 80%, greater than 90%, greaterthan 95%, greater than 98%, greater than 99%, greater than 99.5%, orgreater than 99.9% of the population of mRNA molecules are capped). Insome preferred embodiments, the mRNA used in the methods of the presentinvention has a cap with a cap1 structure, meaning that the 2′ hydroxylof the ribose in the penultimate nucleotide with respect to the capnucleotide is methylated. However, in some embodiments, mRNA used in themethods has a cap with a cap0 structure, meaning that the 2′ hydroxyl ofthe ribose in the penultimate nucleotide with respect to the capnucleotide is not methylated. With some but not all transcripts,transfection of eukaryotic cells with mRNA having a cap with a cap1structure results in a higher level or longer duration of proteinexpression in the transfected cells compared to transfection of the samecells with the same mRNA but with a cap having a cap0 structure. In someembodiments, the mRNA used in the methods of the present invention has amodified cap nucleotide. In some experiments performed prior to theexperiments presented in the EXAMPLES herein, the present applicantsfound that, when 1079 or IMR90 human fibroblast cells were transfectedwith OCT4 mRNA that contained either uridine, or pseudouridine in placeof uridine, the pseudouridine-containing mRNA was translated at a higherlevel or for a longer duration than the mRNA that contained uridine.Therefore, in some preferred embodiments, one or more or all of theuridines contained in the mRNA(s) used in the methods of the presentinvention is/are replaced by pseudouridine (e.g., by substitutingpseudouridine-5′-triphosphate in the IVT reaction to synthesize the RNAin place of uridine-5′-triphosphate). However, in some embodiments, themRNA used in the methods of the invention contains uridine and does notcontain pseudouridine. In some preferred embodiments, the mRNA comprisesat least one modified nucleoside (e.g., selected from the groupconsisting of a pseudouridine (ψ), 5-methylcytosine (m⁵C),5-methyluridine (m⁵U), 2′-O-methyluridine (Um or m^(2′-O)U),2-thiouridine (s²U), and N⁶-methyladenosine (m⁶A)) in place of at leasta portion of the corresponding unmodified canonical nucleoside (e.g., inplace of substantially all of the corresponding unmodified A, C, G, or Tcanonical nucleoside). In some preferred embodiments, the mRNA comprisesat least one modified nucleoside selected from the group consisting of apseudouridine (ψ) and 5-methylcytosine (m⁵C). In some preferredembodiments, the mRNA comprises both pseudouridine (ψ) and5-methylcytosine (m⁵C). In addition, in order to accomplish specificgoals, a nucleic acid base, sugar moiety, or internucleotide linkage inone or more of the nucleotides of the mRNA that is introduced into aeukaryotic cell in any of the methods of the invention may comprise amodified nucleic acid base, sugar moiety, or internucleotide linkage.

The invention is also not limited with respect to the source of the mRNAthat is delivered into the eukaryotic cell in any of the methods of theinvention. In some embodiments, such as those described in the EXAMPLES,the mRNA is synthesized by in vitro transcription of a DNA templatecomprising a gene cloned in a linearized plasmid vector or by in vitrotranscription of a DNA template that is synthesized by PCR or RT-PCR(i.e., by IVT of a PCR amplification product), capping using a cappingenzyme system or by co-transcriptional capping by incorporation of adinucleotide cap analog (e.g., an ARCA) during the IVT, andpolyadenylation using a poly(A) polymerase. In some preferredembodiments, the mRNA is synthesized by IVT of a DNA template comprisinga gene cloned in a linearized plasmid vector or a PCR or RT-PCRamplification product, wherein the DNA template encodes a 3′poly(A)tail. In some other embodiments, the mRNA that is delivered into theeukaryotic cell in any of the methods of the invention is deriveddirectly from a cell or a biological sample. For example, in someembodiments, the mRNA derived from a cell or biological sample isobtained by amplifying the mRNA from the cell or biological sample usingan RNA amplification reaction, and capping the amplified mRNA using acapping enzyme system or by co-transcriptional capping by incorporationof a dinucleotide cap analog (e.g., an ARCA) during the IVT, and, if theamplified mRNA does not already contain a template-encoded poly(A) tailfrom the RNA amplification reaction, polyadenylating the amplified mRNAusing a poly(A) polymerase.

With respect to the methods comprising introducing mRNA encoding one ormore iPS cell induction factors in order to generate a dedifferentiatedcell (e.g., an iPS cell), the invention is not limited by the nature ofthe iPS cell induction factors used. Any mRNA encoding one or moreprotein induction factors now known, or later discovered, that find usein dedifferentiation, are contemplated for use in the present invention.In some embodiments, one or more mRNAs encoding for KLF4, LIN28, c-MYC,NANOG, OCT4, or SOX2 are employed. Oct-3/4 and certain members of theSox gene family (Sox1, Sox2, Sox3, and Sox15) have been identified astranscriptional regulators involved in the induction process. Additionalgenes, however, including certain members of the Klf family (Klf1, Klf2,Klf4, and Klf5), the Myc family (C-myc, L-myc, and N-myc), Nanog, andLIN28, have been identified to increase the induction efficiency. Anyone or more such factors may be used as desired.

While the compositions and methods of the invention may be used togenerate iPS cells, the invention is not limited to the generation ofsuch cells. For example, in some embodiments, mRNA encoding one or morereprogramming factors is introduced into a cell in order to generate acell with a changed state of differentiation compared to the cell intowhich the mRNA was introduced. For example, in some embodiments, mRNAencoding one or more iPS cell induction factors is used to generate adedifferentiated cell that is not an iPS cells. Such cells find use inresearch, drug screening, and other applications.

In some embodiments, the present invention further provides methodsemploying the dedifferentiated cells generated by the above methods. Forexample, such cells find use in research, drug screening, andtherapeutic applications in humans or other animals. For example, insome embodiments, the cells generated find use in the identification andcharacterization of iPS cell induction factors as well as other factorsassociated with differentiation or dedifferentiation. In someembodiments, the generated dedifferentiated cells are transplanted intoan organism or into a tissue residing in vitro or in vivo. In someembodiments, an organism, tissue, or culture system housing thegenerated cells is exposed to a test compound and the effect of the testcompound on the cells or on the organism, tissue, or culture system isobserved or measured.

In some other embodiments, a dedifferentiated cell generated using theabove methods (e.g., an iPS cell) is further treated to generate adifferentiated cell that has the same state of differentiation or celltype compared to the somatic cell from which the dedifferentiated cellwas generated. In some other embodiments, the dedifferentiated cellgenerated using the above methods (e.g., an iPS cell) is further treatedto generate a differentiated cell that has a different state ofdifferentiation or cell type compared to the somatic cell from which thededifferentiated cell was generated. In some embodiments, thedifferentiated cell is generated from the generated dedifferentiatedcell (e.g., the generated iPS cell) by introducing mRNA encoding one ormore reprogramming factors into the generated iPS cell during one ormultiple treatments and maintaining the cell into which the mRNA isintroduced under conditions wherein the cell is viable and isdifferentiated into a cell that has a changed state of differentiationor cell type compared to the generated dedifferentiated cell (e.g., thegenerated iPS cell) into which the mRNA encoding the one or morereprogramming factors is introduced. In some of these embodiments, thegenerated differentiated cell that has the changed state ofdifferentiation is used for research, drug screening, or therapeuticapplications (e.g., in humans or other animals). For example, thegenerated differentiated cells find use in the identification andcharacterization of reprogramming factors associated withdifferentiation. In some embodiments, the generated differentiated cellsare transplanted into an organism or into a tissue residing in vitro orin vivo. In some embodiments, an organism, tissue, or culture systemhousing the generated differentiated cells is exposed to a test compoundand the effect of the test compound on the cells or on the organism,tissue, or culture system is observed or measured.

In some preferred embodiments of the method comprising introducing mRNAencoding one or more iPSC induction factors into a somatic cell andmaintaining the cell under conditions wherein the cell is viable and themRNA that is introduced into the cell is expressed in sufficient amountand for sufficient time to generate a dedifferentiated cell (e.g.,wherein the dedifferentiated cell is an induced pluripotent stem cell),the sufficient time to generate a dedifferentiated cell is less than oneweek. In some preferred embodiments of this method, the reprogrammingefficiency for generating dedifferentiated cells is greater than orequal to 50 dedifferentiated cells (e.g., iPSCs) per 3×10⁵ input cellsinto which the mRNA is introduced. In some preferred embodiments of thismethod, the reprogramming efficiency for generating dedifferentiatedcells is greater than or equal to 100 dedifferentiated cells (e.g.,iPSCs) per 3×10⁵ input cells into which the mRNA is introduced. In somepreferred embodiments of this method, the reprogramming efficiency forgenerating dedifferentiated cells is greater than or equal to 150dedifferentiated cells (e.g., iPSCs) per 3×10⁵ input cells into whichthe mRNA is introduced. In some preferred embodiments of this method,the reprogramming efficiency for generating dedifferentiated cells isgreater than or equal to 200 dedifferentiated cells (e.g., iPSCs) per3×10⁵ input cells into which the mRNA is introduced. In some preferredembodiments of this method, the reprogramming efficiency for generatingdedifferentiated cells is greater than or equal to 300 dedifferentiatedcells (e.g., iPSCs) per 3×10⁵ input cells into which the mRNA isintroduced. In some preferred embodiments of this method, thereprogramming efficiency for generating dedifferentiated cells isgreater than or equal to 400 dedifferentiated cells (e.g., iPSCs) per3×10⁵ input cells into which the mRNA is introduced. In some preferredembodiments of this method, the reprogramming efficiency for generatingdedifferentiated cells is greater than or equal to 500 dedifferentiatedcells (e.g., iPSCs) per 3×10⁵ input cells into which the mRNA isintroduced. In some preferred embodiments of this method, thereprogramming efficiency for generating dedifferentiated cells isgreater than or equal to 600 dedifferentiated cells per 3×10⁵ inputcells (e.g., iPSCs) into which the mRNA is introduced. In some preferredembodiments of this method, the reprogramming efficiency for generatingdedifferentiated cells is greater than or equal to 700 dedifferentiatedcells (e.g., iPSCs) per 3×10⁵ input cells into which the mRNA isintroduced. In some preferred embodiments of this method, thereprogramming efficiency for generating dedifferentiated cells isgreater than or equal to 800 dedifferentiated cells (e.g., iPSCs) per3×10⁵ input cells into which the mRNA is introduced. In some preferredembodiments of this method, the reprogramming efficiency for generatingdedifferentiated cells is greater than or equal to 900 dedifferentiatedcells (e.g., iPSCs) per 3×10⁵ input cells into which the mRNA isintroduced. In some preferred embodiments of this method, thereprogramming efficiency for generating dedifferentiated cells isgreater than or equal to 1000 dedifferentiated cells (e.g., iPSCs) per3×10⁵ input cells into which the mRNA is introduced. Thus, in somepreferred embodiments, this method was greater than 2-fold moreefficient than the published protocol comprising delivery ofreprogramming factors with a viral vector (e.g., a lentivirus vector).In some preferred embodiments, this method was greater than 5-fold moreefficient than the published protocol comprising delivery ofreprogramming factors with a viral vector (e.g., a lentivirus vector).In some preferred embodiments, this method was greater than 10-fold moreefficient than the published protocol comprising delivery ofreprogramming factors with a viral vector (e.g., a lentivirus vector).In some preferred embodiments, this method was greater than 20-fold moreefficient than the published protocol comprising delivery ofreprogramming factors with a viral vector (e.g., a lentivirus vector).In some preferred embodiments, this method was greater than 25-fold moreefficient than the published protocol comprising delivery ofreprogramming factors with a viral vector (e.g., a lentivirus vector).In some preferred embodiments, this method was greater than 30-fold moreefficient than the published protocol comprising delivery ofreprogramming factors with a viral vector (e.g., a lentivirus vector).In some preferred embodiments, this method was greater than 35-fold moreefficient than the published protocol comprising delivery ofreprogramming factors with a viral vector (e.g., a lentivirus vector).In some preferred embodiments, this method was greater than 40-fold moreefficient than the published protocol comprising delivery ofreprogramming factors with a viral vector (e.g., a lentivirus vector).

The present invention further provides compositions (systems, kits,reaction mixtures, cells, mRNA) used or useful in the methods and/orgenerated by the methods described herein. For example, in someembodiments, the present invention provides an mRNA encoding an iPS cellinduction factor, the mRNA having pseudouridine in place of uridine.

The present invention further provides compositions comprising atransfection reagent and an mRNA encoding an iPS cell induction factor(e.g., a mixture of transfection reagent and mRNA). In some embodiments,the compositions comprise mRNA encoding a plurality (e.g., 2 or more, 3or more, 4 or more, 5 or more, or 6) of iPS cell induction factors,including, but not limited to, KLF4, LIN28, c-MYC, NANOG, OCT4, andSOX2.

The compositions may further comprise any other reagent or componentsufficient, necessary, or useful for practicing any of the methodsdescribed herein. Such reagents or components include, but are notlimited to, transfection reagents, culture medium (e.g., MEF-conditionmedium), cells (e.g., somatic cells, iPS cells), containers, boxes,buffers, inhibitors (e.g., RNase inhibitors), labels (e.g., fluorescent,luminescent, radioactive, etc.), positive and/or negative controlmolecules, reagents for generating capped mRNA, dry ice or otherrefrigerants, instructions for use, cell culture equipment,detection/analysis equipment, and the like.

This invention provides RNA, oligoribonucleotide, and polyribonucleotidemolecules comprising pseudouridine or a modified nucleoside, genetherapy vectors comprising same, gene therapy methods and genetranscription silencing methods comprising same, methods of reducing animmunogenicity of same, and methods of synthesizing same.

In one embodiment, the present invention provides a messenger RNAcomprising ⋅a pseudouridine residue. In another embodiment, the presentinvention provides an RNA molecule encoding a protein of interest, saidRNA molecule comprising a pseudouridine residue. In another embodiment,the present invention provides an in vitro-transcribed RNA molecule,comprising a pseudouridine or a modified nucleoside. In anotherembodiment, the present invention provides an in vitro-synthesizedoligoribonucleotide, comprising a pseudouridine or a modifiednucleoside, wherein the modified nucleoside is m⁵C, m⁵U, m⁶A, s²U, ψ, or2′-O-methyl-U. In another embodiment, the present invention provides agene-therapy vector, comprising an in vitro-synthesizedpolyribonucleotide molecule, wherein the polyribonucleotide moleculecomprises a pseudouridine or a modified nucleoside.

In another embodiment, the present invention provides a double-strandedRNA (dsRNA) molecule containing, as part of its sequence, apseudouridine or a modified nucleoside and further comprising an siRNAor shRNA. In another embodiment, the dsRNA molecule is greater than 50nucleotides in length. Each possibility represents a separate embodimentof the present invention.

In another embodiment, the present invention provides a method forinducing a mammalian cell to produce a recombinant protein, comprisingcontacting the mammalian cell with an in vitro-synthesized RNA moleculeencoding the recombinant protein, the in vitro-synthesized RNA moleculecomprising a pseudouridine or a modified nucleoside, thereby inducing amammalian cell to produce a recombinant protein.

In another embodiment, the present invention provides a method fortreating anemia in a subject, comprising contacting a cell of thesubject with an in vitro-synthesized RNA molecule, the invitro-synthesized RNA molecule encoding erythropoietin, thereby treatinganemia in a subject.

In another embodiment, the present invention provides a method fortreating a vasospasm in a subject, comprising contacting a cell of thesubject with an in vitro-synthesized RNA molecule, the invitro-synthesized RNA molecule encoding inducible nitric oxide synthase(iNOS), thereby treating a vasospasm in a subject.

In another embodiment, the present invention provides a method forimproving a survival rate of a cell in a subject, comprising contactingthe cell with an in vitro-synthesized RNA molecule, the invitro-synthesized RNA molecule encoding a heat shock protein, therebyimproving a survival rate of a cell in a subject.

In another embodiment, the present invention provides a method fordecreasing an incidence of a restenosis of a blood vessel following aprocedure that enlarges the blood vessel, comprising contacting a cellof the blood vessel with an in vitro-synthesized RNA molecule, the invitro-synthesized RNA molecule encoding a heat shock protein, therebydecreasing an incidence of a restenosis in a subject.

In another embodiment, the present invention provides a method forincreasing a hair growth from a hair follicle is a scalp of a subject,comprising contacting a cell of the scalp with an in vitro synthesizedRNA molecule, the in vitro-synthesized RNA molecule encoding atelomerase or an immunosuppressive protein, thereby increasing a hairgrowth from a hair follicle.

In another embodiment, the present invention provides a method ofinducing expression of an enzyme with antioxidant activity in a cell,comprising contacting the cell with an in vitro-synthesized RNAmolecule, the in vitro-synthesized RNA molecule encoding the enzyme,thereby inducing expression of an enzyme with antioxidant activity in acell.

In another embodiment, the present invention provides a method fortreating cystic fibrosis in a subject, comprising contacting a cell ofthe subject with an in vitro-synthesized RNA molecule, the invitro-synthesized RNA molecule encoding Cystic Fibrosis TransmembraneConductance Regulator (CFTR), thereby treating cystic fibrosis in asubject.

In another embodiment, the present invention provides a method fortreating an X-linked agammaglobulinemia in a subject, comprisingcontacting a cell of the subject with an in vitro synthesized RNAmolecule, the in vitro-synthesized RNA molecule encoding a Bruton'styrosine kinase, thereby treating an X-linked agammaglobulinemia.

In another embodiment, the present invention provides a method fortreating an adenosine deaminase severe combined immunodeficiency (ADASCID) in a subject, comprising contacting a cell of the subject with anin vitro-synthesized RNA molecule, the in vitro-synthesized RNA moleculeencoding an ADA, thereby treating an ADA SCID.

In another embodiment, the present invention provides a method forproducing a recombinant protein, comprising contacting an in vitrotranslation apparatus with an in vitro-synthesized polyribonucleotide,the in vitro-synthesized polyribonucleotide comprising a pseudouridineor a modified nucleoside, thereby producing a recombinant protein.

In another embodiment, the present invention provides a method ofsynthesizing an in vitro-transcribed RNA molecule comprising a modifiednucleotide with a pseudouridine modified nucleoside, comprisingcontacting an isolated polymerase with a mixture of unmodifiednucleotides and the modified nucleotide.

In another embodiment, the present invention provides an in vitrotranscription apparatus, comprising: an unmodified nucleotide, anucleotide containing a pseudouridine or a modified nucleoside, and apolymerase. In another embodiment, the present invention provides an invitro transcription kit, comprising: an unmodified nucleotide, anucleotide containing a pseudouridine or a modified nucleoside, and apolymerase. Each possibility represents a separate embodiment of thepresent invention.

DESCRIPTION OF THE FIGURES

The following figures form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these figures in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1 shows that mRNAs encoding each of the six human reprogrammingfactors, prepared as described in the EXAMPLES, are translated andlocalized to the predicted subcellular locations after transfection intohuman newborn 1079 fibroblasts. Untreated human 1079 fibroblasts: PhotosA, E, I, M, Q, and U show phase contrast images of the untreated human1079 fibroblasts which were not transfected with an mRNA encoding areprogramming factor and photos B, F, J, N, R, and V show fluorescentimages of the same fields after the cells were stained with an antibodyspecific for each reprogramming factor; these results show that therewas little or none of these endogenous reprogramming factor proteins inuntreated human 1079 fibroblasts. Treated human 1079 fibroblasts: PhotosC, G, K, O, S, and W show phase contrast images of the human 1079fibroblasts which were transfected with an mRNA encoding the indicatedreprogramming factor, and photos D, H, L, P, T, and X show fluorescentimages of the same fields after the cells were stained with an antibodyspecific for each reprogramming factor 24 hours after transfection.These results show that each of the reprogramming factor proteins wasexpressed in the human 1079 fibroblast cells 24 hours after transfectionwith the respective reprogramming factor-encoding mRNAs and that thereprogramming factor proteins were localized in the predictedsubcellular locations. A-T are at 20× magnification. U-X are at 10×magnification.

FIG. 2 shows that mRNA encoding human reprogramming factors (KLF4,LIN28, c-MYC, NANOG, OCT4, and SOX2) produce iPS cells in human somaticcells. FIG. 2 shows bright-field (A, C) and immunofluorescent (B, D)images of an iPS cell colony at 12 days after the final transfectionwith mRNA encoding reprogramming factors. NANOG staining is observed incolony #1 (B, D). Images A and B are at 10× magnification. C and D areat 20× magnification.

FIG. 3 shows that iPS colonies derived from human 1079 and IMR90 somaticcells are positive for NANOG and TRA-1-60. FIG. 3 shows phase contrast(A, D, G) and immunofluorescent (B, C, E, F, H, I) images of iPScolonies derived from 1079 cells (A, D) and IMR90 cells (G). The sameiPS colony shown in (A) is positive for both NANOG (B) and TRA-1-60 (C).The iPS colony shown in (D) is NANOG-positive (E) and TRA-1-60-positive(F). The iPS colony generated from IMR90 fibroblasts (G) is alsopositive for both NANOG (H) and TRA-1-60 (I). All images are at 20×magnification.

FIG. 4 shows that rapid, enhanced-efficiency iPSC colony formation isachieved by transfecting cells with mRNA encoding reprogramming factorsin MEF-conditioned medium. Over 200 colonies were detected 3 days afterthe final transfection; in the 10-cm dish, IMR90 cells were transfectedthree times with 36 μg of each reprogramming mRNA (i.e., encoding KLF4,LIN28, c-MYC, NANOG, OCT4, and SOX2). Representative iPSC colonies areshown at 4× (A, B), 10× (C-E) and 20× magnification (F). Eight daysafter the final mRNA transfection with mRNAs encoding the sixreprogramming factors, more than 1000 iPSC colonies were counted inIMR90 cells transfected with 18 μg (G, I) or 36 μg (H) of each of thesix mRNAs. Representative colonies are shown at 4× magnification (G-H)and at 10× magnification (I).

FIG. 5 shows that 1079- and IMR90-derived iPSC colonies are positive forboth NANOG and TRA-1-60. Eight days after the final mRNA transfectionwith 36 μg of mRNA for each of the six reprogramming factors, the1079-derived iPSC colonies (shown in A, D, and G) are positive for NANOG(B, E, and H) and TRA-1-60 (C, F, and I). Eight days after the finalmRNA transfection with 18 μg (J-L) or 36 μg (M-O) of mRNA for each ofthe six reprogramming factors, IMR90-derived iPS colonies are alsopositive for NANOG (K, N) and TRA-1-60 (L, O).

FIG. 6. Production of TNF-α by MDDCs transfected with natural RNA,demonstrating that unmodified in vitro-synthesized RNA and bacterial RNAand mammalian mitochondrial RNA is highly immunogenic, while othermammalian RNA is weakly immunogenic. Human MDDCs were incubated withLipofectin® alone, or complexed with R-848 (1 μg/ml), or RNA (5 μg/ml)from 293 cells (total, nuclear and cytoplasmic RNAs), mouse heart(polyA+mRNA), human platelet mitochondrial RNA, bovine tRNA, bacterialtRNA and total RNA (E. coli) with or without RNase digestion. After 8 h,TNF-0 was measured in the supernatants by ELISA. Mean values±SEM areshown. Results are representative of 3 independent experiments.

FIGS. 7A-C. TLR-dependent activation by RNA demonstrates that m6A ands2U modification blocks TLR3 signaling, while all modifications blockTLR7 and TLR8 signaling, and that less modified bacterial RNA andunmodified in vitro-transcribed RNA activates all three TLR. (A)Aliquots (1 μg) of in vitro-transcribed RNA-1571 without (none) or withm⁵C, m⁶A, ψ, m⁵U or ST nucleoside modifications were analyzed ondenaturing agarose gel followed by ethidium bromide-staining and UVillumination. (B) 293 cells expressing human TLR3, TLR7, TLR8 andcontrol vectors were treated with Lipofectin® alone, Lipofectin®-R-848(1 μg/ml) or RNA (5 μg/ml). Modified nucleosides present in RNA-730 andRNA-1571 are noted. (C) CpG ODN-2006 (5 μg/ml), LPS (1.0 μg/ml) and RNAisolates were obtained from rat liver, mouse cell line (TUBO) and humanspleen (total), human platelet mitochondrial RNA, or from two differentE. coli sources. 293-hTLR9 cells served as control. After 8 h, IL-8 wasmeasured in the supernatants by ELISA. Mean values±SEM are shown. Celllines containing hTLR3-targeted siRNA are indicated with asterisk. Theresults are representative of four independent experiments.

FIGS. 8A-E. Cytokine production by RNA-transfected DC demonstrates thatall modifications block activation of cytokine generated DC, while onlyuridine modifications block blood-derived DC activation. MDDC generatedwith GM-CSF/IL-4 (A, C) or GM-CSF/IFN-α MDDCs (B), and primary DC1 andDC2 (D) were treated for 8 to 16 h with Lipofectin® alone,Lipofectin®-R-848 (1 μg/ml) or RNA (5 μg/ml). Modified nucleosidespresent in RNA-1571 are noted. TNF-α, IL-12(p70) and IFN-α were measuredin the supernatant by ELISA. Mean values±SEM are shown. The results arerepresentative of 10 (A and C), 4 (B), and 6 (D) independentexperiments. E. Activation of DC by RNA. MDDC were treated for 20 h withLipofectin® alone or complexed with 1 μg/ml poly(I):(C) or R-848 aspositive controls (top panel) or Lipofectin® complexed with theindicated RNA (5 μg/ml; bottom panel). Modified nucleosides present inRNA-1886 are noted. Expression of CD83, CD80, and HLA-DR was determinedby flow cytometry.

FIGS. 9A-B. Activation of DC by RNA demonstrates that all nucleosidemodification inhibits the RNA-mediated DC activation. MDDC were treatedfor 20 h with Lipofectin® alone, Lipofectin®-R-848 (1 μg/ml) orRNA-1571, modified as indicated (5 μg/ml). (A) CD83 and HLA-DR staining.(B) TNF-α levels in the supernatants and mean fluorescence of CD80 andCD86 in response to incubation with RNA. The volume of medium wasincreased 30-fold for flow cytometry, as indicated by the asterisk. Dataare representative of four independent experiments.

FIGS. 10A-C. Capped RNA-1571 containing different amounts (0, 1, 10, 50,90, 99 and 100% of modified nucleoside, relative to the correspondingunmodified NTP) were transcribed, and it was found that modification ofonly a few nucleosides resulted in an inhibition of activation of DC. A.All transcripts were digested to monophosphates and analyzed byreversed-phase HPLC to determine the relative amount of modifiednucleoside incorporation. Representative absorbance profiles obtained atthe indicated (ψ:U) ratios are shown. Elution times are noted for3′-monophosphates of pseudouridine (ψ), cytidine (C), guanosine (G),uridine (U), 7-methylguanosine (“m7G”) and adenosine (“A”). (B) Modifiednucleoside content of RNA-1571. The expected percentage of m⁶A, ψ(pseudouridine), or m⁵C in RNA-1571 was calculated based on the relativeamount of modified NTP in the transcription reaction and the nucleosidecomposition of RNA-1571 (A: 505, U: 451, C: 273, G: 342). Values formeasured modified nucleoside content were determined based onquantitation of the HPLC chromatograms. Notes: A: values (%) for m⁶ATP,ψTP and m⁵CTP relative to ATP, UTP and CTP, respectively. B: values form⁶A, ψ and m⁵C monophosphates relative to all NMPs. (C) MDDC weretransfected with Lipofectin® complexed capped RNA-1571 (5 μg/ml)containing the indicated amount of m⁶A, ψ or m⁵C. After 8 h, TNF-α wasmeasured in the supernatants. Data expressed as relative inhibition ofTNF-α. Mean values±SEM obtained in 3 independent experiments are shown.

FIGS. 11A-C. TNF-α expression by oligoribonucleotide-transfected DCsdemonstrates that as few as one modified nucleoside reduces DCactivation. (A) Sequences of oligoribonucleotides (ORN) synthesizedchemically (ORN1-4) (SEQ ID NOs: 6-9) or transcribed in vitro (ORN5-6)(SEQ ID NOs: 10-11) are shown. Positions of modified nucleosides Um(2′-O-methyluridine), m⁵C and ψ are highlighted. Human MDDC weretransfected with Lipofectin® alone (medium), R-848 (1 μg/ml) orLipofectin® complexed with RNA (5 μg/ml). Where noted, cells weretreated with 2.5 μg/ml cycloheximide (CHX). (B). After 8 h incubation,TNF-α was measured in the supernatant. (C) RNA from the cells wasanalyzed by Northern blot. Representative mean values±SEM of 3independent experiments are shown.

FIGS. 12A-B. A. ψV-modified mRNA does not stimulate pro-inflammatorycytokine production in vivo. Serum samples (6 h after injection) wereanalyzed by ELISA and revealed that 3 μg of unmodified mRNA induced ahigher level of IFN-α than did 3 μg of ψ-modified mRNA (P<0.001). Levelsof IFN-α induced by 3 μg of ψ-modified mRNA were similar to thoseobtained when animals were injected with uncomplexed lipofectin. Valuesare expressed as the mean±s.e.m. (n=3 or 5 animals/group). B. Similarresults were observed with TNF-α.

FIG. 13. mRNA containing pseudouridine (ψ) does not activate PKR. ψ:pseudouridine. Control: unmodified RNA. m5C: mRNA with m⁵C modification.

FIG. 14. Increased expression of luciferase frompseudouridine-containing mRNA in rabbit reticulocyte lysate. Luc-Y: mRNAwith pseudouridine modification; luc-C: unmodified RNA. Data isexpressed by normalizing luciferase activity to unmodified luciferaseRNA.

FIGS. 15A-B. Increased expression of renilla frompseudouridine-containing mRNA in cultured cells. A. 293 cells. B. Murineprimary, bone marrow-derived mouse dendritic cells. renilla-Y: mRNA withpseudouridine modification; renilla-C:unmodified RNA. RNA was modifiedwith m⁵C, m⁶A, and m⁵U as noted.

FIGS. 16A-C. A. Additive effect of 3′ and 5′ elements on translationefficiency of ψ-modified mRNA. 293 cells were transfected with fireflyluciferase conventional and ψ-modified mRNAs that had 5′ cap (capLuc),50 nt-long 3′ polyA-tail (TEVlucA50), both or neither of these elements(capTEVlucA50 and Luc, respectively). Cells were lysed 4 h later andluciferase activities measured in aliquots ( 1/20th) of the totallysates. B. ψ-modified mRNA is more stable than unmodified mRNA. 293cells transfected with capTEVlucA_(n) containing unmodified orψ-modified nucleosides were lysed at the indicated times followingtransfection. Aliquots ( 1/20th) of the lysates were assayed forluciferase. Standard errors are too small to be visualized with errorbars. C. Expression of β-galactosidase is enhanced using ψ-modified mRNAcompared with conventional mRNA. 293 cells seeded in 96-well plates weretransfected with lipofectin-complexed mRNAs (0.25 μg/well) encodingbacterial β-galactosidase (lacZ). The transcripts had cap and 3′polyA-tail that were either 30 nt-long (caplacZ) or ˜200 nt-long(caplacZ-An). Constructs made using conventional U or nucleosides weretested. Cells were fixed and stained with X-gal, 24 h post-transfection.Images were taken by inverted microscopy (40 and 100× magnification)from representative wells.

FIGS. 17A-D. A. Expression of renilla following intracerebral injectionof modified or unmodified encoding mRNA. Rat brain cortex was injectedat 8 sites/animals. One hemisphere was injected with capped,renilla-encoding RNA with pseudouridine modification (capRenilla-Y),while the corresponding hemisphere with capped RNA with no nucleosidemodification (capRenilla-C). Data from 2 animals (6 injection sites) areshown. BG; lower level of detection of the assay. B.Intravenously-delivered ψ-modified mRNA is expressed in spleen.Lipofectin-complexed ψmRNA (0.3 μg capTEVlucAn/mouse) was administeredby tail vein injection. Animals were sacrificed at 2 and 4 hpost-injection and luciferase activities measured in aliquots ( 1/10th)of organs homogenized in lysis buffer. Values represent luciferaseactivities in the whole organs. C. ψ-modified mRNA exhibits greaterstability and translation in vivo. Lipofectin-complexed capTEVlucAn (0.3μg/60 μl/animal) with or without ψ modifications was delivered i.v. tomice. Animals were sacrificed at 1, 4 and 24 h post-injection, and ½ oftheir spleens were processed for luciferase enzyme measurements (leftpanel) and the other half for RNA analyses (right panel). Luciferaseactivities were measured in aliquots (⅕th) of the homogenate made fromhalf of the spleens. Plotted values represent luciferase activities inthe whole spleen and are expressed as the mean±s.e.m. (n=3 or 4/point).D. Expression of firefly luciferase following intratracheal injection ofmRNA. capTEVluc-Y: capped, firefly luciferase encodingpseudouridine-modified RNA. CapTEVluc-C: capped RNA with no nucleosidemodification.

FIG. 18. Protein production is dependent on the amount of mRNA deliveredintravenously in mice. The indicated amounts of lipofectin-complexednucleic acids, capTEVlucAn mRNA with or without ψ constituents andpCMVluc plasmid DNA in a volume of 60 μl/animal were delivered by i.v.injection into mice. Animals injected with mRNA or plasmid DNA weresacrificed at 6 h or 24 h post-injection, respectively, and luciferaseactivities were measured in aliquots ( 1/10th) of their spleenshomogenized in lysis buffer. The value from each animal is shown, andshort horizontal lines indicate the mean; N.D., not detectable.

FIG. 19. Expression of firefly luciferase following intratrachealdelivery of encoding mRNA. mRNA were complexed to lipofectin (or PEI, asnoted) and animals were injected with 0.3 μg firefly luciferase-encodingmRNA with or without ψ modification, then sacrificed 3 hours later.Lungs were harvested and homogenized, and luciferase activity wasmeasured in aliquots of the lysed organs.

FIG. 20. ψ-modified mRNA does not induce inflammatory mediators afterpulmonary delivery. Induction of TNF-α and IFN-α in serum followingintratracheal delivery of luciferase-encoding unmodified mRNA orψ-modified mRNA. Serum levels of TNF-α and IFN-α were determined byELISA 24 hours after mRNA delivery.

FIGS. 21A-C show the results from Example 35: Firefly or Renillaluciferase encoding mRNA with the indicated modifications were complexedto lipofectin and delivered to murine dendritic (A) and HEK293T (B)cells. Human DC were transfected with firefly or renillaluciferase-encoding mRNA complexed with TransIT with the indicatedmodifications (C). Data is expressed as the fold-change compared tounmodified mRNA.

FIG. 22 shows the results from Example 36: T7 polymerase transcriptionreactions used for the generation of mRNA results in large quantities ofRNA of the correct size, but also contains contaminants. This isvisualized by application of RNA to a reverse phase HPLC column thatseparates RNA based on size under denaturing conditions. ψ-modifiedTEV-luciferase-A51 RNA was applied to the HPLC column in 38% Buffer Band subjected to a linear gradient of increasing Buffer B to 55%. Theprofile demonstrated both smaller than expected and larger than expectedcontaminants.

FIGS. 23A-B show the results from Example 37: (A) EPO encoding mRNA withthe indicated modifications and with or without HPLC purification weredelivered to murine DCs and EPO levels in the supernatant were measured24 hr later. While m5C/ψ-modified mRNA had the highest level oftranslation prior to HPLC purification, ψ-modified mRNA had the highesttranslation after HPLC purification. (B) Human DCs were transfected withrenilla encoding mRNA with the indicated modifications with or withoutHPLC purification.

FIGS. 24A-C shows the results from Example 38: (A) Human DCs weretransfected with RNA complexed to TransIT with the indicatedmodifications with or without HPLC purification. IFN-α levels weremeasured after 24 hr. HPLC purification increased the immunogenicity ofunmodified RNA, which is dependent of the sequence, as other unmodifiedRNAs had similar levels of IFN-α or reduced levels after HPLCpurification. ψ-modified RNA had unmeasurable levels of IFN-α, similarto control treated DCs. (B) ψ-modified RNA before (−) and after HPLCpurification (P1 and P2) was analyzed for dsRNA using dot blotting witha monoclonal antibody specific for dsRNA (J2). Purification of RNAremoved dsRNA contamination. (C) ψ-modified RNA encoding iPS factors areimmunogenic, which is removed by HPLC purification of the RNA.

FIG. 25 provides the mRNA coding sequence for KLF4 (SEQ ID NO:12) andLIN28 (SEQ ID NO:13).

FIG. 26 provides the mRNA coding sequence for cMYC (SEQ ID NO:14) andNANOG (SEQ ID NO:15).

FIG. 27 provides the mRNA coding sequence for OCT4 (SEQ ID NO:16) andSOX2 (SEQ ID NO:17).

FIG. 28 shows that mRNA encoding human reprogramming factors (KLF4,c-MYC, OCT4, and SOX2) produce iPS cells in primary human keratinocytecells. FIG. 28 shows phase contrast images of HEKn cells at 2 days (A)and iPS colony formation at 11 days (B) and 20 days (C) after the finaltransfection with mRNA encoding 4 reprogramming factors. Images are at10× magnification.

FIG. 29 shows that mRNA encoding human reprogramming factors (KLF4,LIN28, c-MYC, NANOG, OCT4, and SOX2) produce iPS cells in humankeratinocytes that are positive for known iPS cell markers. FIG. 29shows phase contrast images of colonies derived from HEKn cells (A, D,and G). The same iPS colony shown in (A) is positive for both KLF4 (B)and LIN28 (C). The iPS colony shown in (D) is SSEA4-positive (E) andTRA-1-60-positive (F). The iPS colony shown in (G) is NANOG-positive(H). All images are at 20× magnification.

FIG. 30 shows increases in the expression of 3 iPS-associated messagesin HEKn cells transfected with 4 reprogramming mRNAs (KLF4, c-MYC, OCT4,and SOX2) which did not include the reprogramming factor NANOG.Increased expression of the messages was detected by qPCR and isnormalized to GAPDH expression. The expression level of each message isdepicted in relation to the level found in the original cell line.

DEFINITIONS

The present invention will be understood and interpreted based on termsas defined below.

As used herein “substantially all,” in reference to single-strandedcomplete mRNAs comprising a pseudouridine or 5-methylcytidine residue,means that of all the single-stranded complete mRNAs present in asample, at least 95% have either a pseudouridine or 5-methylcytidineresidue.

As used herein “essentially all,” in reference to single-strandedcomplete mRNAs comprising a pseudouridine or 5-methylcytidine residue,means that of all the single-stranded complete mRNAs present in asample, at least 99% have either a pseudouridine or 5-methylcytidineresidue.

As used herein “RNA contaminant molecules” are molecules that compriseRNA residues and that can at least partially activate an immune responsewhen transfected into a cell (e.g., by activating RNA sensors such asRNA-dependent protein kinase (PKR), retinoic acid-inducible gene-I(RIG-I), Toll-like receptor (TLR)3, TLR7, TLR8, and oligoadenylatesynthetase (OAS), or RNA molecules that can at least partially activatean RNA interference (RNAi) response (e.g., including a response to largedouble-stranded RNA molecules or to small double-stranded RNA molecules(siRNAs)) in the cell. Exemplary RNA contaminant molecules include, butare not limited to: partial or non-full-length mRNAs encoding only aportion of a reprogramming factor (e.g., a non-full-length iPS cellinduction factor); single-stranded mRNAs that are greater than thefull-length mRNA that encodes a reprogramming factor (e.g., an iPS cellinduction factor), e.g., without being bound by theory, by “run-on IVT”or other mechanisms; double-stranded large or small mRNA molecules; anduncapped mRNA molecules.

As used herein, a purified RNA preparation is “substantially free” ofRNA contaminant molecules (or a particular recited RNA contaminant),when less than 0.5% of the total RNA in the purified RNA preparationconsists of RNA contaminant molecules (or a particularly recited RNAcontaminant). The amounts and relative amounts of non-contaminant mRNAmolecules and RNA contaminant molecules (or a particular RNAcontaminant) may be determined by HPLC or other methods used in the artto separate and quantify RNA molecules.

As used herein, a purified RNA preparation is “essentially free” of RNAcontaminant molecules (or a particular recited RNA contaminant), whenless than 1.0% of the total RNA in the purified RNA preparation consistsof RNA contaminant molecules (or a particularly recited RNAcontaminant). The amounts and relative amounts of non-contaminant mRNAmolecules and RNA contaminant molecules (or a particular RNAcontaminant) may be determined by HPLC or other methods used in the artto separate and quantify RNA molecules.

As used herein, a purified RNA preparation is “virtually free” of RNAcontaminant molecules (or a particular recited RNA contaminant), whenless than 0.1% of the total RNA in the purified RNA preparation consistsof RNA contaminant molecules (or a particularly recited RNAcontaminant). The amounts and relative amounts of non-contaminant mRNAmolecules and RNA contaminant molecules (or a particular RNAcontaminant) may be determined by HPLC or other methods used in the artto separate and quantify RNA molecules.

As used herein, a purified RNA preparation is “free” of RNA contaminantmolecules (or a particular recited RNA contaminant), when less than0.01% of the total RNA in the purified RNA preparation consists of RNAcontaminant molecules (or a particularly recited RNA contaminant). Theamounts and relative amounts of non-contaminant mRNA molecules and RNAcontaminant molecules (or a particular RNA contaminant) may bedetermined by HPLC or other methods used in the art to separate andquantify RNA molecules.

The terms “comprising”, “containing”, “having”, “include”, and“including” are to be construed as “including, but not limited to”unless otherwise noted. The terms “a,” “an,” and “the” and similarreferents in the context of describing the invention and, specifically,in the context of the appended claims, are to be construed to cover boththe singular and the plural unless otherwise noted. The use of any andall examples or exemplary language (“for example”, “e.g.”, “such as”) isintended merely to illustrate aspects or embodiments of the invention,and is not to be construed as limiting the scope thereof, unlessotherwise claimed.

With respect to the use of the word “derived”, such as for an RNA(including mRNA) or a polypeptide that is “derived” from a sample,biological sample, cell, tumor, or the like, it is meant that the RNA orpolypeptide either was present in the sample, biological sample, cell,tumor, or the like, or was made using the RNA in the sample, biologicalsample, cell, tumor, or the like by a process such as an in vitrotranscription reaction, or an RNA amplification reaction, wherein theRNA or polypeptide is either encoded by or a copy of all or a portion ofthe RNA or polypeptide molecules in the original sample, biologicalsample, cell, tumor, or the like. By way of example, such RNA can befrom an in vitro transcription or an RNA amplification reaction, with orwithout cloning of cDNA, rather than being obtained directly from thesample, biological sample, cell, tumor, or the like, so long as theoriginal RNA used for the in vitro transcription or an RNA amplificationreaction was from the sample, biological sample, cell, tumor, or thelike. The terms “sample” and “biological sample” are used in theirbroadest sense and encompass samples or specimens obtained from anysource that contains or may contain eukaryotic cells, includingbiological and environmental sources. As used herein, the term “sample”when used to refer to biological samples obtained from organisms,includes bodily fluids (e.g., blood or saliva), feces, biopsies, swabs(e.g., buccal swabs), isolated cells, exudates, and the like. Theorganisms include fungi, plants, animals, and humans. However, theseexamples are not to be construed as limiting the types of samples ororganisms that find use with the present invention. In addition, inorder to perform research or study the results related to use of amethod or composition of the invention, in some embodiments, a “sample”or “biological sample” comprises fixed cells, treated cells, celllysates, and the like. In some embodiments, such as embodiments of themethod wherein the mRNA is delivered into a cell from an organism thathas a known disease or into a cell that exhibits a disease state or aknown pathology, the “sample” or “biological sample” also comprisesbacteria or viruses.

As used herein, the term “incubating” and variants thereof meancontacting one or more components of a reaction with another componentor components, under conditions and for sufficient time such that adesired reaction product is formed.

As used herein, a “nucleoside” consists of a nucleic acid base (e.g.,the canonical nucleic acid bases: guanine (G), adenine (A), thymine (T),uracil (U), and cytosine (C)); or a modified nucleic acid base (e.g.,5-methylcytosine (m⁵C)), that is covalently linked to a pentose sugar(e.g., ribose or 2′-deoxyribose), whereas and a “nucleotide” or“mononucleotide” consists of a nucleoside that is phosphorylated at oneof the hydroxyl groups of the pentose sugar. Linear nucleic acidmolecules are said to have a “5′ terminus” (5′ end) and a “3′ terminus”(3′ end) because, except with respect to capping or adenylation (e.g.,adenylation by a ligase), mononucleotides are joined in one directionvia a phosphodiester linkage to make oligonucleotides orpolynucleotides, in a manner such that a phosphate on the 5′ carbon ofone mononucleotide sugar moiety is joined to an oxygen on the 3′ carbonof the sugar moiety of its neighboring mononucleotide. Therefore, an endof a linear single-stranded oligonucleotide or polynucleotide or an endof one strand of a linear double-stranded nucleic acid (RNA or DNA) isreferred to as the “5′ end” if its 5′ phosphate is not joined or linkedto the oxygen of the 3′ carbon of a mononucleotide sugar moiety, and asthe “3′ end” if its 3′ oxygen is not joined to a 5′ phosphate that isjoined to a sugar of another mononucleotide. A terminal nucleotide, asused herein, is the nucleotide at the end position of the 3′ or 5′terminus.

In order to accomplish specific goals, a nucleic acid base, sugarmoiety, or internucleoside (or internucleotide) linkage in one or moreof the nucleotides of the mRNA that is introduced into a eukaryotic cellin any of the methods of the invention may comprise a modified base,sugar moiety, or internucleoside linkage. For example, in addition tothe other modified nucleotides discussed elsewhere herein for performingthe methods of the present invention, one or more of the nucleotides ofthe mRNA can also have a modified nucleic acid base comprising orconsisting of: xanthine; allyamino-uracil; allyamino-thymidine;hypoxanthine; 2-aminoadenine; 5-propynyl uracil; 5-propynyl cytosine;4-thiouracil; 6-thioguanine; an aza or deaza uracil; an aza or deazathymidine; an aza or deaza cytosines; an aza or deaza adenine; or an azaor deaza guanines; or a nucleic acid base that is derivatized with abiotin moiety, a digoxigenin moiety, a fluorescent or chemiluminescentmoiety, a quenching moiety or some other moiety in order to accomplishone or more specific other purposes; and/or one or more of thenucleotides of the mRNA can have a sugar moiety, such as, but notlimited to: 2′-fluoro-2′-deoxyribose or 2′-O-methyl-ribose, whichprovide resistance to some nucleases; or 2′-amino-2′-deoxyribose or2′-azido-2′-deoxyribose, which can be labeled by reacting them withvisible, fluorescent, infrared fluorescent or other detectable dyes orchemicals having an electrophilic, photoreactive, alkynyl, or otherreactive chemical moiety.

In some embodiments of the invention, one or more of the nucleotides ofthe mRNA comprises a modified internucleoside linkage, such as aphosphorothioate, phosphorodithioate, phosphoroselenate, orphosphorodiselenate linkage, which are resistant to some nucleases,including in a dinucleotide cap analog (Grudzien-Nogalska et al. 2007)that is used in an IVT reaction for co-transcriptional capping of theRNA, or in the poly(A) tail (e.g., by incorporation of a nucleotide thathas the modified phosphorothioate, phosphorodithioate,phosphoroselenate, or phosphorodiselenate linkage during IVT of the RNAor, e.g., by incorporation of ATP that contains the modifiedphosphorothioate, phosphorodithioate, phosphoroselenate, orphosphorodiselenate linkage into a poly(A) tail on the RNA bypolyadenylation using a poly(A) polymerase). The invention is notlimited to the modified nucleic acid bases, sugar moieties, orinternucleoside linkages listed, which are presented to show exampleswhich may be used for a particular purpose in a method.

As used herein, a “nucleic acid” or a “polynucleotide” or an“oligonucleotide” is a covalently linked sequence of nucleotides inwhich the 3′ position of the sugar moiety of one nucleotide is joined bya phosphodiester bond to the 5′ position of the sugar moiety of the nextnucleotide (i.e., a 3′ to 5′ phosphodiester bond), and in which thenucleotides are linked in specific sequence; i.e., a linear order ofnucleotides. In some embodiments, the nucleic acid or polynucleotide oroligonucleotide consists of or comprises 2′-deoxyribonucleotides (DNA).In some embodiments, the oligonucleotide consists of or comprisesribonucleotides (RNA).

The terms “isolated” or “purified” when used in relation to apolynucleotide or nucleic acid, as in “isolated RNA” or “purified RNA”refers to a nucleic acid that is identified and separated from at leastone contaminant with which it is ordinarily associated in its source.Thus, an isolated or purified nucleic acid (e.g., DNA and RNA) ispresent in a form or setting different from that in which it is found innature, or a form or setting different from that which existed prior tosubjecting it to a treatment or purification method. For example, agiven DNA sequence (e.g., a gene) is found on the host cell chromosometogether with other genes as well as structural and functional proteins,and a specific RNA (e.g., a specific mRNA encoding a specific protein),is found in the cell as a mixture with numerous other RNAs and othercellular components. The isolated or purified polynucleotide or nucleicacid may be present in single-stranded or double-stranded form.

A “cap” or a “cap nucleotide” means a nucleoside-5′-triphosphate that,under suitable reaction conditions, is used as a substrate by a cappingenzyme system and that is thereby joined to the 5′-end of an uncappedRNA comprising primary RNA transcripts or RNA having a 5′-diphosphate.The nucleotide that is so joined to the RNA is also referred to as a“cap nucleotide” herein. A “cap nucleotide” is a guanine nucleotide thatis joined through its 5′ end to the 5′ end of a primary RNA transcript.The RNA that has the cap nucleotide joined to its 5′ end is referred toas “capped RNA” or “capped RNA transcript” or “capped transcript.” Acommon cap nucleoside is 7-methylguanosine or N⁷-methylguanosine(sometimes referred to as “standard cap”), which has a structuredesignated as “m⁷G,” in which case the capped RNA or “m⁷G-capped RNA”has a structure designated as m⁷G(5′)ppp(5′)N₁(pN)_(x)-OH(3′), or moresimply, as m⁷GpppN₁(pN)_(x) or m⁷G[5′]ppp[5′]N, wherein m⁷G representsthe 7-methylguanosine cap nucleoside, ppp represents the triphosphatebridge between the 5′ carbons of the cap nucleoside and the firstnucleotide of the primary RNA transcript, N₁(pN)_(x)-OH(3′) representsthe primary RNA transcript, of which N₁ is the most 5′-nucleotide, “p”represents a phosphate group, “G” represents a guanosine nucleoside,“m⁷” represents the methyl group on the 7-position of guanine, and“[5′]” indicates the position at which the “p” is joined to the riboseof the cap nucleotide and the first nucleoside of the mRNA transcript(“N”). In addition to this “standard cap,” a variety of othernaturally-occurring and synthetic cap analogs are known in the art. RNAthat has any cap nucleotide is referred to as “capped RNA.” The cappedRNA can be naturally occurring from a biological sample or it can beobtained by in vitro capping of RNA that has a 5′ triphosphate group orRNA that has a 5′ diphosphate group with a capping enzyme system (e.g.,vaccinia capping enzyme system or Saccharomyces cerevisiae cappingenzyme system). Alternatively, the capped RNA can be obtained by invitro transcription (IVT) of a DNA template that contains an RNApolymerase promoter, wherein, in addition to the GTP, the IVT reactionalso contains a dinucleotide cap analog (e.g., a m⁷GpppG cap analog oran N⁷-methyl, 2′-O-methyl-GpppG ARCA cap analog or an N⁷-methyl,3′-O-methyl-GpppG ARCA cap analog) using methods known in the art (e.g.,using an AMPLICAP™ T7 capping kit or a MESSAGEMAX™ T7 ARCA-CAPPEDMESSAGE Transcription Kit, EPICENTRE or CellScript).

Capping of a 5′-triphosphorylated primary mRNA transcript in vivo (orusing a capping enzyme system in vitro) occurs via several enzymaticsteps (Higman et al. 1992, Martin et al. 1975, Myette and Niles 1996).

The following enzymatic reactions are involved in capping of eukaryoticmRNA:

(1) RNA triphosphatase cleaves the 5′-triphosphate of mRNA to adiphosphate, pppN₁(p)N_(x)-OH(3′)→ppN₁(pN)_(x)-OH(3′)+Pi; and then

(2) RNA guanyltransferase catalyzes joining of GTP to the 5′-diphosphateof the most 5′ nucleotide (N₁) of the mRNA,ppN₁(pN)_(x)-OH(3′)+GTP→G(5′)ppp(5′)N₁(pN)_(x)-OH(3′)+PPi; and finally,

(3) guanine-7-methyltransferase, using S-adenosyl-methionine (AdoMet) asa co-factor, catalyzes methylation of the 7-nitrogen of guanine in thecap nucleotide,G(5′)ppp(5′)N₁(pN)_(x)-OH(3′)+AdoMet→m⁷G-(5′)ppp(5′)N₁(pN)_(x)-OH(3′)+AdoHyc.RNA that results from the action of the RNA triphosphatase and the RNAguanyltransferase enzymatic activities, as well as RNA that isadditionally methylated by the guanine-7-methyltransferase enzymaticactivity, is referred to herein as “5′ capped RNA” or “capped RNA”, anda “capping enzyme system” or, more simply, a “capping enzyme” hereinmeans any combination of one or more polypeptides having the enzymaticactivities that result in “capped RNA.” Capping enzyme systems,including cloned forms of such enzymes, have been identified andpurified from many sources and are well known in the art (Banerjee 1980,Higman et al. 1992, Higman et al. 1994, Myette and Niles 1996, Shuman1995, Shuman 2001, Shuman et al. 1980, Wang et al. 1997). Any cappingenzyme system that can convert uncapped RNA that has a 5′ polyphosphateto capped RNA can be used to provide a capped RNA for any of theembodiments of the present invention. In some embodiments, the cappingenzyme system is a poxvirus capping enzyme system. In some preferredembodiments, the capping enzyme system is vaccinia virus capping enzyme.In some embodiments, the capping enzyme system is Saccharomycescerevisiae capping enzyme. Also, in view of the fact that genes encodingRNA triphosphatase, RNA guanyltransferase andguanine-7-methyltransferase from one source can complement deletions inone or all of these genes from another source, the capping enzyme systemcan originate from one source, or one or more of the RNA triphosphatase,RNA guanyltransferase, and/or guanine-7-methyltransferase activities cancomprise a polypeptide from a different source.

A “modified cap nucleotide” of the present invention means a capnucleotide wherein the sugar, the nucleic acid base, or theinternucleoside linkage is chemically modified compared to thecorresponding canonical 7-methylguanosine cap nucleotide. Examples of amodified cap nucleotide include a cap nucleotide comprising: (i) amodified 2′- or 3′-deoxyguanosine-5′-triphosphate (or guanine 2′- or3′-deoxyribonucleic acid-5′-triphosphate) wherein the 2′- or 3′-deoxyposition of the deoxyribose sugar moiety is substituted with a groupcomprising an amino group, an azido group, a fluorine group, a methoxygroup, a thiol (or mercapto) group or a methylthio (or methylmercapto)group; or (ii) a modified guanosine-5′-triphosphate, wherein the O⁶oxygen of the guanine base is methylated; or (iii) 3′-deoxyguanosine.For the sake of clarity, it will be understood herein that an“alkoxy-substituted deoxyguanosine-5′-triphosphate” can also be referredto as an “O-alkyl-substituted guanosine-5′-triphosphate”; by way ofexample, but without limitation,2′-methoxy-2′-deoxyguanosine-5′-triphosphate (2′-methoxy-2′-dGTP) and3′-methoxy-3′-deoxyguanosine-5′-triphosphate (3′-methoxy-3′-dGTP) canalso be referred to herein as 2′-O-methylguanosine-5′-triphosphate(2′-OMe-GTP) and 3′-O-methylguanosine-5′-triphosphate (3′-OMe-GTP),respectively. Following joining of the modified cap nucleotide to the5′-end of the uncapped RNA comprising primary RNA transcripts (or RNAhaving a 5′-diphosphate), the portion of said modified cap nucleotidethat is joined to the uncapped RNA comprising primary RNA transcripts(or RNA having a 5′-diphosphate) may be referred to herein as a“modified cap nucleoside” (i.e., without referring to the phosphategroups to which it is joined), but sometimes it is referred to as a“modified cap nucleotide”.

A “modified-nucleotide-capped RNA” is a capped RNA molecule that issynthesized using a capping enzyme system and a modified cap nucleotide,wherein the cap nucleotide on its 5′ terminus comprises the modified capnucleotide, or a capped RNA that is synthesize co-transcriptionally inan in vitro transcription reaction that contains a modified dinucleotidecap analog wherein the dinucleotide cap analog contains the chemicalmodification in the cap nucleotide. In some embodiments, the modifieddinucleotide cap analog is an anti-reverse cap analog or ARCA (Grudzienet al. 2004, Jemielity et al. 2003, Grudzien-Nogalska et al. 2007, Penget al. 2002, Stepinski et al. 2001).

A “primary RNA” or “primary RNA transcript” means an RNA molecule thatis synthesized by an RNA polymerase in vivo or in vitro and which RNAmolecule has a triphosphate on the 5′-carbon of its most 5′ nucleotide.

An “RNA amplification reaction” or an “RNA amplification method” means amethod for increasing the amount of RNA corresponding to one or multipledesired RNA sequences in a sample. For example, in some embodiments, theRNA amplification method comprises: (a) synthesizing first-strand cDNAcomplementary to the one or more desired RNA molecules by RNA-dependentDNA polymerase extension of one or more primers that anneal to thedesired RNA molecules; (b) synthesizing double-stranded cDNA from thefirst-strand cDNA using a process wherein a functional RNA polymerasepromoter is joined thereto; and (c) contacting the double-stranded cDNAwith an RNA polymerase that binds to said promoter under transcriptionconditions whereby RNA corresponding to the one or more desired RNAmolecules is obtained. Unless otherwise stated related to a specificembodiment of the invention, an RNA amplification reaction according tothe present invention means a sense RNA amplification reaction, meaningan RNA amplification reaction that synthesizes sense RNA (e.g., RNAhaving the same sequence as an mRNA or other primary RNA transcript,rather than the complement of that sequence). Sense RNA amplificationreactions known in the art, which are encompassed within this definitioninclude, but are not limited to, the methods which synthesize sense RNAdescribed in Ozawa et al. (Ozawa et al. 2006) and in U.S. PatentApplication Nos. 20090053775; 20050153333; 20030186237; 20040197802; and20040171041. The RNA amplification method described in U.S. PatentApplication No. 20090053775 is a preferred method for obtainingamplified RNA derived from one or more cells, which amplified RNA isthen used to make mRNA for use in the methods of the present invention.

A “poly-A polymerase” (“PAP”) means a template-independent RNApolymerase found in most eukaryotes, prokaryotes, and eukaryotic virusesthat selectively uses ATP to incorporate AMP residues to 3′-hydroxylatedends of RNA. Since PAP enzymes that have been studied from plants,animals, bacteria and viruses all catalyze the same overall reaction(Edmonds 1990) are highly conserved structurally (Gershon 2000) and lackintrinsic specificity for particular sequences or sizes of RNA moleculesif the PAP is separated from proteins that recognize AAUAAApolyadenylation signals (Wilusz and Shenk 1988), purified wild-type andrecombinant PAP enzymes from any of a variety of sources can be used forthe present invention. In some embodiments, a PAP enzyme fromSaccharomyces (e.g., from S. cerevisiae) is used for polyadenylation tomake purified RNA preparations comprising or consisting of one or moremodified mRNAs, each of which encodes a reprogramming factor (e.g., aniPS cell induction factor). In some embodiments, a PAP enzyme from E.coli is used for polyadenylation to make purified RNA preparationscomprising or consisting of one or more modified mRNAs, each of whichencodes a reprogramming factor (e.g., an iPS cell induction factor).

A “reprogramming factor” means a protein, polypeptide, or otherbiomolecule that, when used alone or in combination with other factorsor conditions, causes a change in the state of differentiation of a cellin which the reprogramming factor is introduced or expressed. In somepreferred embodiments of the methods of the present invention, thereprogramming factor is a protein or polypeptide that is encoded by anmRNA that is introduced into a cell, thereby generating a cell thatexhibits a changed state of differentiation compared to the cell inwhich the mRNA was introduced. In some preferred embodiments of themethods of the present invention, the reprogramming factor is atranscription factor. One embodiment of a reprogramming factor used in amethod of the present invention is an “iPS cell induction factor.”

An “iPS cell induction factor” or “iPSC induction factor” is a protein,polypeptide, or other biomolecule that, when used alone or incombination with other reprogramming factors, causes the generation ofiPS cells from somatic cells. Examples of iPS cell induction factorsinclude OCT4, SOX2, c-MYC, KLF4, NANOG and LIN28. iPS cell inductionfactors include full length polypeptide sequences or biologically activefragments thereof. Likewise an mRNA encoding an iPS cell inductionfactor may encode a full length polypeptide or biologically activefragments thereof. The mRNA coding sequence for exemplary iPS inductionfactors are shown in FIG. 25 (KLF4 and LIN28), 26 (cMYC and NANOG), and27 (OCT4 and SOX2). In certain embodiments, the present inventionemploys the sequences or similar sequences shown in these figures,including mRNA molecules that additionally comprise, joined to thesemRNA sequences, oligoribonucleotides which exhibit any of the 5′ and 3′UTR sequences, Kozak sequences, IRES sequences, cap nucleotides, and/orpoly(A) sequences used in the experiments described herein, or which aregenerally known in the art and which can be used in place of those usedherein by joining them to these protein-coding mRNA sequences for thepurpose of optimizing translation of the respective mRNA molecules inthe cells and improving their stability in the cell in order toaccomplish the methods described herein.

“Differentiation” or “cellular differentiation” means the naturallyoccurring biological process by which a cell that exhibits a lessspecialized state of differentiation or cell type (e.g., a fertilizedegg cell, a cell in an embryo, or a cell in a eukaryotic organism)becomes a cell that exhibits a more specialized state of differentiationor cell type. Scientists, including biologists, cell biologists,immunologists, and embryologists, use a variety of methods and criteriato define, describe, or categorize different cells according to their“cell type,” “differentiated state,” or “state of differentiation.” Ingeneral, a cell is defined, described, or categorized with respect toits “cell type,” “differentiated state,” or “state of differentiation”based on one or more phenotypes exhibited by that cell, which phenotypescan include shape, a biochemical or metabolic activity or function, thepresence of certain biomolecules in the cell (e.g., based on stains thatreact with specific biomolecules), or on the cell (e.g., based onbinding of one or more antibodies that react with specific biomoleculeson the cell surface). For example, in some embodiments, different celltypes are identified and sorted using a cell sorter orfluorescent-activated cell sorter (FACS) instrument. “Differentiation”or “cellular differentiation” can also occur to cells in culture.

The term “reprogramming” as used herein means differentiation orcellular differentiation that occurs in response to delivery of one ormore reprogramming factors into the cell, directly (e.g., by delivery ofprotein or polypeptide reprogramming factors into the cell) orindirectly (e.g., by delivery of the purified RNA preparation of thepresent invention which comprises one or more mRNA molecules, each ofwhich encodes a reprogramming factor) and maintaining the cells underconditions (e.g., medium, temperature, oxygen and CO₂ levels, matrix,and other environmental conditions) that are conducive fordifferentiation. The term “reprogramming” when used herein is notintended to mean or refer to a specific direction or path ofdifferentiation (e.g., from a less specialized cell type to a morespecialized cell type) and does not exclude processes that proceed in adirection or path of differentiation than what is normally observed innature. Thus, in different embodiments of the present invention,“reprogramming” means and includes any and all of the following:

-   -   (1) “Dedifferentiation”, meaning a process of a cell that        exhibits a more specialized state of differentiation or cell        type (e.g., a mammalian fibroblast, a keratinocyte, a muscle        cell, or a neural cell) going to a cell that exhibits a less        specialized state of differentiation or cell type (e.g., an iPS        cell);    -   (2) “Transdifferentiation”, meaning a process of a cell that        exhibits a more specialized state of differentiation or cell        type (e.g., a mammalian fibroblast, a keratinocyte, or a neural        cell) going to another more specialized state of differentiation        or cell type (e.g., from a fibroblast or keratinocyte to a        muscle cell); and    -   (3) “Redifferentiation” or “Expected Differentiation” or Natural        Differentiation”, meaning a process of a cell that exhibits any        particular state of differentiation or cell type going to        another state of differentiation or cell type as would be        expected in nature if the cell was present in its natural place        and environment (e.g., in an embryo or an organism), whether        said process occurs in vivo in an organism or in culture (e.g.,        in response to one or more reprogramming factors).

DESCRIPTION OF THE INVENTION

The present invention provides compositions and methods forreprogramming the state of differentiation of eukaryotic cells,including human or other animal cells, by contacting the cells withpurified RNA preparations comprising or consisting of one or moredifferent single-strand mRNA molecules that each encode a reprogrammingfactor (e.g., an iPS cell induction factor). The purifiedsingle-stranded mRNA molecules preferably comprise at least one modifiednucleoside selected from the group consisting of a pseudouridine (ψ),5-methylcytosine (m⁵C), 5-methyluridine (m⁵U), 2′-O-methyluridine (Um orm^(2′-O) U), 2-thiouridine (s²U), and N⁶-methyladenosine (m⁶A) in placeof at least a portion (e.g., including substantially all) of thecorresponding unmodified canonical nucleoside of the correspondingunmodified A, C, G, or T canonical nucleoside. In addition, thesingle-stranded mRNA molecules are preferably purified to besubstantially free of RNA contaminant molecules that would activate anunintended response, decrease expression of the single-stranded mRNA,and/or activate RNA sensors in the cells. In certain embodiments, thepurified RNA preparations are substantially free of RNA contaminantmolecules that are: shorter or longer than the full-lengthsingle-stranded mRNA molecules, double-stranded, and/or uncapped RNA. Insome preferred embodiments, the invention provides compositions andmethods for reprogramming differentiated eukaryotic cells, includinghuman or other animal somatic cells, by contacting the cells withpurified RNA preparations comprising or consisting of one or moredifferent single-strand mRNA molecules that each encode an iPS cellinduction factor.

In certain embodiments, the mRNA used in the purified RNA preparationsis purified to remove substantially, essentially, or virtually all ofthe contaminants, including substantially, essentially, or virtually allof the RNA contaminants. The present invention is not limited withrespect to the purification methods used to purify the mRNA, and theinvention includes use of any method that is known in the art ordeveloped in the future in order to purify the mRNA and removecontaminants, including RNA contaminants, that interfere with theintended use of the mRNA. For example, in preferred embodiments, thepurification of the mRNA removes contaminants that are toxic to thecells (e.g., by inducing an innate immune response in the cells, or, inthe case of RNA contaminants comprising double-stranded RNA, by inducingRNA interference (RNAi), e.g., via siRNA or long RNAi molecules) andcontaminants that directly or indirectly decrease translation of themRNA in the cells). In some embodiments, the mRNA is purified by HPLCusing a method described herein, including in the Examples. In certainembodiments, the mRNA is purified using on a polymeric resin substratecomprising a C18 derivatized styrene-divinylbenzene copolymer and atriethylamine acetate (TEAA) ion pairing agent is used in the columnbuffer along with the use of an acetonitrile gradient to elute the mRNAand separate it from the RNA contaminants in a size-dependent manner; insome embodiments, the mRNA purification is performed using HPLC, but insome other embodiments a gravity flow column is used for thepurification. In some embodiments, the mRNA is purified using a methoddescribed in the book entitled “RNA Purification and Analysis” byDouglas T. Gjerde, Lee Hoang, and David Hornby, published by Wiley-VCH,2009, herein incorporated by reference. In some embodiments, the mRNApurification is carried out in a non-denaturing mode (e.g., at atemperature less than about 50 degrees C., e.g., at ambienttemperature). In some embodiments, the mRNA purification is carried outin a partially denaturing mode (e.g., at a temperature less than about50 degrees C. and 72 degrees C.). In some embodiments, the mRNApurification is carried out in a denaturing mode (e.g., at a temperaturegreater than about 72 degrees C.). Of course, those with knowledge inthe art will know that the denaturing temperature depends on the meltingtemperature (Tm) of the mRNA that is being purified as well as on themelting temperatures of RNA, DNA, or RNA/DNA hybrids which contaminatethe mRNA. In some other embodiments, the mRNA is purified as describedby Mellits K H et al. (Removal of double-stranded contaminants from RNAtranscripts: synthesis of adenovirus VA RNA1 from a T7 vector. NucleicAcids Research 18: 5401-5406, 1990, herein incorporated by reference inits entirety). These authors used a three step purification to removethe contaminants which may be used in embodiments of the presentinvention. Step 1 was 8% polyacrylamide gel electrophoresis in 7M urea(denaturing conditions). The major RNA band was excised from the gelslice and subjected to 8% polyacrylamide gel electrophoresis undernondenaturing condition (no urea) and the major band recovered from thegel slice. Further purification was done on a cellulose CF-11 columnusing an ethanol-salt buffer mobile phase which separates doublestranded RNA from single stranded RNA (Franklin R M. 1966. Proc. Natl.Acad. Sci. USA 55: 1504-1511; Barber R. 1966. Biochem. Biophys. Acta114:422; and Zelcer A et al. 1982. J. Gen. Virol. 59: 139-148, all ofwhich are herein incorporated by reference) and the final purificationstep was cellulose chromatography. In some other embodiments, the mRNAis purified using an hydroxylapatite (HAP) column under eithernon-denaturing conditions or at higher temperatures (e.g., as describedby Pays E. 1977. Biochem. J. 165: 237-245; Lewandowski L J et al. 1971.J. Virol. 8: 809-812; Clawson G A and Smuckler E A. 1982. CancerResearch 42: 3228-3231; and/or Andrews-Pfannkoch C et al. 2010. Appliedand Environmental Microbiology 76: 5039-5045, all of which are hereinincorporated by reference). In some other embodiments, the mRNA ispurified by weak anion exchange liquid chromatography undernon-denaturing conditions (e.g., as described by Easton L E et al. 2010.RNA 16: 647-653 to clean up in vitro transcription reactions, hereinincorporated by reference). In some embodiments, the mRNA is purifiedusing a combination of any of the above methods or another method knownin the art or developed in the future. In still another embodiment, themRNA used in the compositions and methods of the present invention ispurified using a process which comprises treating the mRNA with anenzyme that specifically acts (e.g., digests) one or more contaminantRNA or contaminant nucleic acids (e.g., including DNA), but which doesnot act on (e.g., does not digest) the desired mRNA. For example, insome embodiments, the mRNA used in the compositions and methods of thepresent invention is purified using a process which comprises treatingthe mRNA with a ribonuclease III (RNase III) enzyme (e.g., E. coli RNaseIII) and the mRNA is then purified away from the RNase III digestionproducts. A ribonuclease III (RNase III) enzyme herein means an enzymethat digests double-stranded RNA greater than about twelve basepairs toshore double-stranded RNA fragments. In some embodiments, the mRNA usedin the compositions and methods of the present invention is purifiedusing a process which comprises treating the mRNA with one or more otherenzymes that specifically digest one or more contaminant RNAs orcontaminant nucleic acids (e.g., including DNA).

This invention provides RNA, oligoribonucleotide, and polyribonucleotidemolecules comprising pseudouridine or a modified nucleoside, genetherapy vectors comprising same, gene therapy methods and genetranscription silencing methods comprising same, methods of reducing animmunogenicity of same, and methods of synthesizing same. These modifiedsequences are preferably present in the purified RNA preparationsdescribed herein.

In one embodiment, the present invention provides a messenger RNAcomprising a pseudouridine residue. In another embodiment, the messengerRNA encodes a protein of interest. Each possibility represents aseparate embodiment of the present invention. In another embodiment, thepresent invention provides an RNA molecule encoding a protein ofinterest, said RNA molecule comprising a pseudouridine residue. Inanother embodiment, the present invention provides in vitro-transcribedRNA molecule, comprising a pseudouridine. In another embodiment, thepresent invention provides an in vitro-transcribed RNA molecule,comprising a modified nucleoside.

As provided herein, the present invention provides methods forsynthesizing in vitro-transcribed RNA molecules, comprisingpseudouridine and/or modified nucleosides. In another embodiment, thepresent invention provides a messenger RNA molecule comprising apseudouridine residue.

In another embodiment, an in vitro-transcribed RNA molecule of methodsand compositions of the present invention is synthesized by T7 phage RNApolymerase. In another embodiment, the molecule is synthesized by SP6phage RNA polymerase. In another embodiment, the molecule is synthesizedby T3 phage RNA polymerase. In another embodiment, the molecule issynthesized by a polymerase selected from the above polymerases. Inanother embodiment, the in vitro-transcribed RNA molecule is anoligoribonucleotide. In another embodiment, the in vitro-transcribed RNAmolecule is a polyribonucleotide. Each possibility represents a separateembodiment of the present invention. In another embodiment, the presentinvention provides an in vitro-synthesized oligoribonucleotide,comprising a pseudouridine or a modified nucleoside, wherein themodified nucleoside is m⁵C, m⁵U, m⁶A, s²U, ψ, or 2′-O-methyl-U. Inanother embodiment, the present invention provides an invitro-synthesized polyribonucleotide, comprising a pseudouridine or amodified nucleoside, wherein the modified nucleoside is m⁵C, m⁵U, m⁶A,s²U, ψ or 2′-O-methyl-U.

In another embodiment, the in vitro-synthesized oligoribonucleotide orpolyribonucleotide is a short hairpin (sh)RNA. In another embodiment,the in vitro-synthesized oligoribonucleotide is a small interfering RNA(siRNA). In another embodiment, the in vitro-synthesizedoligoribonucleotide is any other type of oligoribonucleotide known inthe art. Each possibility represents a separate embodiment of thepresent invention.

In another embodiment, an RNA, oligoribonucleotide, orpolyribonucleotide molecule of methods and compositions of the presentinvention further comprises an open reading frame that encodes afunctional protein. In another embodiment, the RNA molecule oroligoribonucleotide molecule functions without encoding a functionalprotein (e.g. in transcriptional silencing), as an RNzyme, etc. Eachpossibility represents a separate embodiment of the present invention.

In another embodiment, the RNA, oligoribonucleotide, orpolyribonucleotide molecule further comprises a poly-A tail. In anotherembodiment, the RNA, oligoribonucleotide, or polyribonucleotide moleculedoes not comprise a poly-A tail. Each possibility represents a separateembodiment of the present invention.

In another embodiment, the RNA, oligoribonucleotide, orpolyribonucleotide molecule further comprises an m7GpppG cap. In anotherembodiment, the RNA, oligoribonucleotide, or polyribonucleotide moleculedoes not comprise an m7GpppG cap. Each possibility represents a separateembodiment of the present invention.

In another embodiment, the RNA, oligoribonucleotide, orpolyribonucleotide molecule further comprises a cap-independenttranslational enhancer. In another embodiment, the RNA,oligoribonucleotide, or polyribonucleotide molecule molecule does notcomprise a cap-independent translational enhancer. In anotherembodiment, the cap-independent translational enhancer is a tobacco etchvirus (TEV) cap-independent translational enhancer. In anotherembodiment, the cap-independent translational enhancer is any othercap-independent translational enhancer known in the art. Eachpossibility represents a separate embodiment of the present invention.

In another embodiment, the present invention provides a gene-therapyvector, comprising an in vitro-synthesized polyribonucleotide molecule,wherein the polyribonucleotide molecule comprises a pseudouridine or amodified nucleoside.

In another embodiment, an RNA, oligoribonucleotide, orpolyribonucleotide molecule of methods and compositions of the presentinvention comprises a pseudouridine. In another embodiment, the RNAmolecule or oligoribonucleotide molecule comprises a modifiednucleoside. In another embodiment, the RNA molecule oroligoribonucleotide molecule is an in vitro-synthesized RNA molecule oroligoribonucleotide. Each possibility represents a separate embodimentof the present invention.

“Pseudouridine” refers, in another embodiment, to m¹acp³ψ(1-methyl-3-(3-amino-3-carboxypropyl) pseudouridine. In anotherembodiment, the term refers to m¹ψ (1-methylpseudouridine). In anotherembodiment, the term refers to ψm (2′-O-methylpseudouridine. In anotherembodiment, the term refers to m⁵D (5-methyldihydrouridine). In anotherembodiment, the term refers to m³ψ (3-methylpseudouridine). In anotherembodiment, the term refers to a pseudouridine moiety that is notfurther modified. In another embodiment, the term refers to amonophosphate, diphosphate, or triphosphate of any of the abovepseudouridines. In another embodiment, the term refers to any otherpseudouridine known in the art. Each possibility represents a separateembodiment of the present invention.

In another embodiment, an RNA, oligoribonucleotide, orpolyribonucleotide molecule of methods and compositions of the presentinvention is a therapeutic oligoribonucleotide.

In another embodiment, the present invention provides a method fordelivering a recombinant protein to a subject, the method comprising thestep of contacting the subject with an RNA, oligoribonucleotide,polyribonucleotide molecule, or a gene-therapy vector of the presentinvention, thereby delivering a recombinant protein to a subject.

In another embodiment, the present invention provides a double-strandedRNA (dsRNA) molecule comprising a pseudouridine or a modified nucleosideand further comprising an siRNA or short hairpin RNA (shRNA). In anotherembodiment, the dsRNA molecule is greater than 50 nucleotides in length.Each possibility represents a separate embodiment of the presentinvention.

In another embodiment, the pseudouridine or a modified nucleoside iswithin the siRNA sequence. In another embodiment, the pseudouridine or amodified nucleoside is outside the siRNA sequence. In anotherembodiment, 1 or more pseudouridine and/or a modified nucleosideresidues are present both within and outside the siRNA sequence. Eachpossibility represents a separate embodiment of the present invention.

In another embodiment, the siRNA or shRNA is contained internally in thedsRNA molecule. In another embodiment, the siRNA or shRNA is containedon one end of the dsRNA molecule. In another embodiment, one or moresiRNA or shRNA is contained on one end of the dsRNA molecule, whileanother one or more is contained internally. Each possibility representsa separate embodiment of the present invention.

In another embodiment, the length of an RNA, oligoribonucleotide, orpolyribonucleotide molecule (e.g. a single-stranded RNA (ssRNA) or dsRNAmolecule) of methods and compositions of the present invention isgreater than 30 nucleotides in length. In another embodiment, the RNAmolecule or oligoribonucleotide is greater than 35 nucleotides inlength. In another embodiment, the length is at least 40 nucleotides. Inanother embodiment, the length is at least 45 nucleotides. In anotherembodiment, the length is at least 55 nucleotides. In anotherembodiment, the length is at least 60 nucleotides. In anotherembodiment, the length is at least 60 nucleotides. In anotherembodiment, the length is at least 80 nucleotides. In anotherembodiment, the length is at least 90 nucleotides. In anotherembodiment, the length is at least 100 nucleotides. In anotherembodiment, the length is at least 120 nucleotides. In anotherembodiment, the length is at least 140 nucleotides. In anotherembodiment, the length is at least 160 nucleotides. In anotherembodiment, the length is at least 180 nucleotides. In anotherembodiment, the length is at least 200 nucleotides. In anotherembodiment, the length is at least 250 nucleotides. In anotherembodiment, the length is at least 300 nucleotides. In anotherembodiment, the length is at least 350 nucleotides. In anotherembodiment, the length is at least 400 nucleotides. In anotherembodiment, the length is at least 450 nucleotides. In anotherembodiment, the length is at least 500 nucleotides. In anotherembodiment, the length is at least 600 nucleotides. In anotherembodiment, the length is at least 700 nucleotides. In anotherembodiment, the length is at least 800 nucleotides. In anotherembodiment, the length is at least 900 nucleotides. In anotherembodiment, the length is at least 1000 nucleotides. In anotherembodiment, the length is at least 1100 nucleotides. In anotherembodiment, the length is at least 1200 nucleotides. In anotherembodiment, the length is at least 1300 nucleotides. In anotherembodiment, the length is at least 1400 nucleotides. In anotherembodiment, the length is at least 1500 nucleotides. In anotherembodiment, the length is at least 1600 nucleotides. In anotherembodiment, the length is at least 1800 nucleotides. In anotherembodiment, the length is at least 2000 nucleotides. In anotherembodiment, the length is at least 2500 nucleotides. In anotherembodiment, the length is at least 3000 nucleotides. In anotherembodiment, the length is at least 4000 nucleotides. In anotherembodiment, the length is at least 5000 nucleotides. Each possibilityrepresents a separate embodiment of the present invention.

In another embodiment, a dsRNA molecule of methods and compositions ofthe present invention is manufactured by in vitro-transcription. Inanother embodiment, the step of in vitro-transcription utilizes T7 phageRNA polymerase. In another embodiment, the in vitro-transcriptionutilizes SP6 phage RNA polymerase. In another embodiment, the invitro-transcription utilizes T3 phage RNA polymerase. In anotherembodiment, the in vitro-transcription utilizes an RNA polymeraseselected from the above polymerases. In another embodiment, the invitro-transcription utilizes any other RNA polymerase known in the art.Each possibility represents a separate embodiment of the presentinvention.

In another embodiment, the dsRNA molecule is capable of being processedby a cellular enzyme to yield the siRNA or shRNA. In another embodiment,the cellular enzyme is an endonuclease. In another embodiment, thecellular enzyme is Dicer. Dicer is an RNase III-family nuclease thatinitiates RNA interference (RNAi) and related phenomena by generation ofthe small RNAs that determine the specificity of these gene silencingpathways (Bernstein E, Caudy A A et al, Role for a bidentateribonuclease in the initiation step of RNA interference. Nature 2001;409(6818): 363-6). In another embodiment, the cellular enzyme is anyother cellular enzyme known in the art that is capable of cleaving adsRNA molecule. Each possibility represents a separate embodiment of thepresent invention.

In another embodiment, the dsRNA molecule contains two siRNA or shRNA.In another embodiment, the dsRNA molecule contains three siRNA or shRNA.In another embodiment, the dsRNA molecule contains more than three siRNAor shRNA. In another embodiment, the siRNA and/or shRNA are liberatedfrom the dsRNA molecule by a cellular enzyme. Each possibilityrepresents a separate embodiment of the present invention.

In another embodiment, the present invention provides a method foradministering an siRNA or shRNA to a cell, comprising administering adsRNA molecule of the present invention, wherein the cell processes thedsRNA molecule to yield the siRNA or shRNA, thereby administering asiRNA or shRNA to a cell.

In another embodiment, the nucleoside that is modified in an RNA,oligoribonucleotide, or polyribonucleotide molecule of methods andcompositions of the present invention is uridine (U). In anotherembodiment, the modified nucleoside is cytidine (C). In anotherembodiment, the modified nucleoside is adenosine (A). In anotherembodiment the modified nucleoside is guanosine (G). Each possibilityrepresents a separate embodiment of the present invention.

In another embodiment, the modified nucleoside of methods andcompositions of the present invention is m⁵C (5-methylcytidine). Inanother embodiment, the modified nucleoside is m⁵U (5-methyluridine). Inanother embodiment, the modified nucleoside is m⁶A (N⁶-methyladenosine).In another embodiment, the modified nucleoside is s²U (2-thiouridine).In another embodiment, the modified nucleoside is ψ(pseudouridine). Inanother embodiment, the modified nucleoside is Um (2′-O-methyluridine).

In other embodiments, the modified nucleoside is m¹A(1-methyladenosine); m²A (2-methyladenosine); Am(2′-O-methyladenosine);ms² m^(G)A (2-methylthio-N⁶-methyladenosine);i^(G)A(˜isopentenyladenosine); ms^(z)i6A(2-methyIthio-N⁶isopentenyladenosine); io⁶A(N⁶-(cis-hydroxyisopentenyl)adenosine); ms²io⁶A(2-methylthio-N⁶⁻(cis-hydroxyisopentenyl)adenosine); g⁶A(N⁶-glycinylcarbamoyladenosine); t⁶A (N⁶-threonylcarbamoyladenosine);ms²t⁶A (2-methylthio-N⁶-threony1carbamoyladenosine); m⁶t⁶A (N⁶-methy1-N⁶-threonylcarbamoyladenosine);hn⁶A(N⁶hydroxynorvalylcarbamoyladenosine); ms²hn⁶A(2-methylthio-N⁶-hydroxynorvalyl carbamoyladenosine); Ar(p)(2′-O-ribosyladenosine (phosphate)); I (inosine); m¹I (1-methylinosine);m¹Im (1,2′-O-dimethylinosine); m³C (3-methylcytidine); Cm(2′-O-methylcytidine); S²C (2thiocytidine); ac⁴C (N⁴-acetylcytidine);f⁵C (5-formylcytidine); m⁵Cm (5,2′-O-dimethylcytidine); ac⁴Cm(N⁴-acetyl-2′-O-methylcytidine); k²C (lysidine); m¹G(1-methylguanosine); m²G (N²-methylguanosine); m⁷G (7-methylguanosine);Gm (2′-O-methylguanosine); m² ₂G (N²,N²-dimethylguanosine); m²Gm(N²,2′-O-dimethylguanosine); m² ₂Gm (N²,N²,2′-O-trimethylguanosine);Gr(p) (2′-O-ribosylguanosine (phosphate)); yW (wybutosine); o₂yW(peroxywybutosine); OHyW (hydroxywybutosine); OHyW* (undermodifiedhydroxywybutosine); imG (wyosine); mimG (methylwyosine); Q (queuosine);oQ (epoxyqueuosine); galQ (galactosyl-queuosine); manQ(mannosylqueuosine); preQ₀ (7-cyano-7-deazaguanosine); preQ₁(7-aminomethyl-7-deazaguanosine); (archaeosine); D (dihydrouridine);m⁵Um (5,2′-O-dimethyluridine); S⁴U (4-thiouridine); m⁵s²U(5-methyl-2-thiouridine); s²Um (2-thio-2′-O-methyluridine); acp³U(3-(3-amino-3-carboxypropyl)uridine); ho⁵U (5-hydroxyuridine); mo⁵U(5-methoxyuridine); cmo⁵U (uridine 5-oxyacetic acid); mcmo⁵U (uridine5-oxyacetic acid methyl ester); chm⁵U(5-(carboxyhydroxymethyl)uridine)); mchm⁵U(5(carboxyhydroxymethyl)uridine methyl ester); mcm⁵U(5-methoxycarbonylmethyluridine); mcm⁵Um(5-methoxycarbonylmethyl-2′-O-methyluridine); mcm⁵s²U(5-methoxycarbonylmethyl-2-thiouridine); nm⁵s²U(5-aminomethyl-2-thiouridine); mnm⁵U (5-methylaminomethyluridine);mnm⁵s²U (5-methylaminomethyl-2-thiouridine); mnmse²U(5-methylaminomethyl-2-selenouridine); ncm⁵U (5-carbamoylmethyluridine);ncm⁵Um (5-carbamoylmethyl-2′-O-methyluridine); cmnm⁵U(5-carboxymethylaminomethyluridine); cmnm⁵Um(5-carboxymethylaminomethyl-2′-methyluridine); cmnm⁵s²U(5-carboxymethylaminomethyl-2-thiouridine); m⁶ ₂ A(N⁶,N⁶-dimethyladenosine); Im (2′-O-methylinosine); m⁴C(N⁴-methylcytidine); m⁴Cm (N⁴,2′-O-dimethylcytidine); hm⁵C(5-hydroxymethy 1 cytidine); m³U (3-methyluridine); cm⁵U(5-carboxymethyluridine); m⁶Am, (N⁶,2′-Odimethyladenosine); m⁶ ₂ Am(N⁶,N⁶, 2′-O-trimethyladenosine); m^(2,7)G(N²,7-dimethylguanosine);m^(2,2,7)G (N²,N²,7-trimethylguanosine); m³Um (3,2′-O-dimethyluridine);m⁵D (5-methyldihydrouridine); f⁵Cm (5-formyl-2′-O-methylcytidine); m¹Gm(1,2′-O-dimethylguanosine); m¹Am (1,2′-O-dimethyladenosine); τm⁵U(5-taurinomethyluridine); τm5s2U (5-taurinomethyl-2-thiouridine));imG-14 (4-demethylwyosine); imG2 (isowyosine); or ac⁶A(N⁶-acetyladenosine). Each possibility represents a separate embodimentof the present invention.

In another embodiment, an RNA, oligoribonucleotide, orpolyribonucleotide molecule of methods and compositions of the presentinvention comprises a combination of 2 or more of the abovemodifications. In another embodiment, the RNA molecule oroligoribonucleotide molecule comprises a combination of 3 or more of theabove modifications. In another embodiment, the RNA molecule oroligoribonucleotide molecule comprises a combination of more than 3 ofthe above modifications. Each possibility represents a separateembodiment of the present invention.

In another embodiment, between 0.1% and 100% of the residues in the RNA,oligoribonucleotide, or polyribonucleotide molecule of methods andcompositions of the present invention are modified (e.g. either by thepresence of pseudouridine or a modified nucleoside base). In anotherembodiment, 0.1% of the residues are modified. In another embodiment,0.2%. In another embodiment, the fraction is 0.3%. In anotherembodiment, the fraction is 0.4%. In another embodiment, the fraction is0.5%. In another embodiment, the fraction is 0.6%. In anotherembodiment, the fraction is 0.8%. In another embodiment, the fraction is1%. In another embodiment, the fraction is 1.5%. In another embodiment,the fraction is 2%. In another embodiment, the fraction is 2.5%. Inanother embodiment, the fraction is 3%. In another embodiment, thefraction is 4%. In another embodiment, the fraction is 5%. In anotherembodiment, the fraction is 6%. In another embodiment, the fraction is8%. In another embodiment, the fraction is 10%. In another embodiment,the fraction is 12%. In another embodiment, the fraction is 14%. Inanother embodiment, the fraction is 16%. In another embodiment, thefraction is 18%. In another embodiment, the fraction is 20%. In anotherembodiment, the fraction is 25%. In another embodiment, the fraction is30%. In another embodiment, the fraction is 35%. In another embodiment,the fraction is 40%. In another embodiment, the fraction is 45%. Inanother embodiment, the fraction is 50%. In another embodiment, thefraction is 60%. In another embodiment, the fraction is 70%. In anotherembodiment, the fraction is 80%. In another embodiment, the fraction is90%. In another embodiment, the fraction is 100%.

In another embodiment, the fraction is less than 5%. In anotherembodiment, the fraction is less than 3%. In another embodiment, thefraction is less than 1%. In another embodiment, the fraction is lessthan 2%. In another embodiment, the fraction is less than 4%. In anotherembodiment, the fraction is less than 6%. In another embodiment, thefraction is less than 8%. In another embodiment, the fraction is lessthan 10%. In another embodiment, the fraction is less than 12%. Inanother embodiment, the fraction is less than 15%. In anotherembodiment, the fraction is less than 20%. In another embodiment, thefraction is less than 30%. In another embodiment, the fraction is lessthan 40%. In another embodiment, the fraction is less than 50%. Inanother embodiment, the fraction is less than 60%. In anotherembodiment, the fraction is less than 70%.

In another embodiment, 0.1% of the residues of a given nucleotide(uridine, cytidine, guanosine, or adenine) are modified. In anotherembodiment, the fraction of the nucleotide is 0.2%. In anotherembodiment, the fraction is 0.3%. In another embodiment, the fraction is0.4%. In another embodiment, the fraction is 0.5%. In anotherembodiment, the fraction is 0.6%. In another embodiment, the fraction is0.8%. In another embodiment, the fraction is 1%. In another embodiment,the fraction is 1.5%. In another embodiment, the fraction is 2%. Inanother embodiment, the fraction is 2.5%. In another embodiment, thefraction is 3%. In another embodiment, the fraction is 4%. In anotherembodiment, the fraction is 5%. In another embodiment, the fraction is6%. In another embodiment, the fraction is 8%. In another embodiment,the fraction is 10%. In another embodiment, the fraction is 12%. Inanother embodiment, the fraction is 14%. In another embodiment, thefraction is 16%. In another embodiment, the fraction is 18%. In anotherembodiment, the fraction is 20%. In another embodiment, the fraction is25%. In another embodiment, the fraction is 30%. In another embodiment,the fraction is 35%. In another embodiment, the fraction is 40%. Inanother embodiment, the fraction is 45%. In another embodiment, thefraction is 50%. In another embodiment, the fraction is 60%. In anotherembodiment, the fraction is 70%. In another embodiment, the fraction is80%. In another embodiment, the fraction is 90%. In another embodiment,the fraction is 100%.

In another embodiment, the fraction of the given nucleotide is less than8%. In another embodiment, the fraction is less than 10%. In anotherembodiment, the fraction is less than 5%. In another embodiment, thefraction is less than 3%. In another embodiment, the fraction is lessthan 1%. In another embodiment, the fraction is less than 2%. In anotherembodiment, the fraction is less than 4%. In another embodiment, thefraction is less than 6%. In another embodiment, the fraction is lessthan 12%. In another embodiment, the fraction is less than 15%. Inanother embodiment, the fraction is less than 20%. In anotherembodiment, the fraction is less than 30%. In another embodiment, thefraction is less than 40%. In another embodiment, the fraction is lessthan 50%. In another embodiment, the fraction is less than 60%. Inanother embodiment, the fraction is less than 70%.

In another embodiment, the terms “ribonucleotide,”“oligoribonucleotide,” and “polyribonucleotide” refers to a string of atleast 2 base-sugar-phosphate combinations. The term includes, in anotherembodiment, compounds comprising nucleotides in which the sugar moietyis ribose. In another embodiment, the term includes both RNA and RNAderivatives in which the backbone is modified. “Nucleotides” refers, inanother embodiment, to the monomeric units of nucleic acid polymers. RNAmay be, in another embodiment, in the form of a tRNA (transfer RNA),snRNA (small nuclear RNA), rRNA (ribosomal RNA), mRNA (messenger RNA),antisense RNA, small interfering RNA (siRNA), micro RNA (miRNA) andribozymes. The use of siRNA and miRNA has been described (Caudy A A etal, Genes & Devel 16: 2491-96 and references cited therein). Inaddition, these forms of RNA may be single, double, triple, or quadruplestranded. The term also includes, in another embodiment, artificialnucleic acids that may contain other types of backbones but the samebases. In another embodiment, the artificial nucleic acid is a PNA(peptide nucleic acid). PNA contain peptide backbones and nucleotidebases and are able to bind, in another embodiment, to both DNA and RNAmolecules. In another embodiment, the nucleotide is oxetane modified. Inanother embodiment, the nucleotide is modified by replacement of one ormore phosphodiester bonds with a phosphorothioate bond. In anotherembodiment, the artificial nucleic acid contains any other variant ofthe phosphate backbone of native nucleic acids known in the art. The useof phosphothiorate nucleic acids and PNA are known to those skilled inthe art, and are described in, for example, Neilsen P E, Curr OpinStruct Biol 9:353-57˜and Raz N K et al Biochem Biophys Res Commun.297:1075-84. The production and use of nucleic acids is known to thoseskilled in art and is described, for example, in Molecular Cloning,(2001), Sambrook and Russell, eds. and Methods in Enzymology: Methodsfor molecular cloning in eukaryotic cells (2003) Purchio and G. C.Fareed. Each nucleic acid derivative represents a separate embodiment ofthe present invention

In another embodiment, the term “oligoribonucleotide” refers to a stringcomprising fewer than 25 nucleotides (nt). In another embodiment,“oligoribonucleotide” refers to a string of fewer than 24 nucleotides.In another embodiment, “oligoribonucleotide” refers to a string of fewerthan 23 nucleotides. In another embodiment, “oligoribonucleotide” refersto a string of fewer than 22 nucleotides. In another embodiment,“oligoribonucleotide” refers to a string of fewer than 21 nucleotides.In another embodiment, “oligoribonucleotide” refers to a string of fewerthan 20 nucleotides. In another embodiment, “oligoribonucleotide” refersto a string of fewer than 19 nucleotides. In another embodiment,“oligoribonucleotide” refers to a string of fewer than 18 nucleotides.In another embodiment, “oligoribonucleotide” refers to a string of fewerthan 17 nucleotides. In another embodiment, “oligoribonucleotide” refersto a string of fewer than 16 nucleotides. Each possibility represents aseparate embodiment of the present invention.

In another embodiment, the term “polyribonucleotide” refers to a stringcomprising more than 25 nucleotides (nt). In another embodiment,“polyribonucleotide” refers to a string of more than 26 nucleotides. Inanother embodiment, “polyribonucleotide” refers to a string of more than28 nucleotides. In another embodiment, “the term” refers to a string ofmore than 30 nucleotides. In another embodiment, “the term” refers to astring of more than 32 nucleotides. In another embodiment, “the term”refers to a string of more than 35 nucleotides. In another embodiment,“the term” refers to a string of more than 40 nucleotides. In anotherembodiment, “the term” refers to a string of more than 50 nucleotides.In another embodiment, “the term” refers to a string of more than 60nucleotides. In another embodiment, “the term” refers to a string ofmore than 80 nucleotides. In another embodiment, “the term” refers to astring of more than 100 nucleotides. In another embodiment, “the term”refers to a string of more than 120 nucleotides. In another embodiment,“the term” refers to a string of more than 150 nucleotides. In anotherembodiment, “the term” refers to a string of more than 200 nucleotides.In another embodiment, “the term” refers to a string of more than 300nucleotides. In another embodiment, “the term” refers to a string ofmore than 400 nucleotides. In another embodiment, “the term” refers to astring of more than 500 nucleotides. In another embodiment, “the term”refers to a string of more than 600 nucleotides. In another embodiment,“the term” refers to a string of more than 800 nucleotides. In anotherembodiment, “the term” refers to a string of more than 1000 nucleotides.In another embodiment, “the term” refers to a string of more than 1200nucleotides. In another embodiment, “the term” refers to a string ofmore than 1400 nucleotides. In another embodiment, “the term” refers toa string of more than 1600 nucleotides. In another embodiment, “theterm” refers to a string of more than 1800 nucleotides. In anotherembodiment, “the term” refers to a string of more than 2000 nucleotides.Each possibility represents a separate embodiment of the presentinvention.

In another embodiment, the present invention provides a method forinducing a mammalian cell to produce a protein of interest, comprisingcontacting the mammalian cell with an in vitro-synthesized RNA moleculeencoding the recombinant protein, the in vitro-synthesized RNA moleculecomprising a pseudouridine or a modified nucleoside, thereby inducing amammalian cell to produce a protein of interest. In another embodiment,the protein of interest is a recombinant protein. Each possibilityrepresents a separate embodiment of the present invention.

“Encoding” refers, in another embodiment, to an RNA molecule thatencodes the protein of interest. In another embodiment, the RNA moleculecomprises an open reading frame that encodes the protein of interest. Inanother embodiment, one or more other proteins is also encoded. Inanother embodiment, the protein of interest is the only protein encoded.Each possibility represents a separate embodiment of the presentinvention.

In another embodiment, the present invention provides a method ofinducing a mammalian cell to produce a recombinant protein, comprisingcontacting the mammalian cell with an in vitro-transcribed RNA moleculeencoding the recombinant protein, the in vitro-transcribed RNA moleculefurther comprising a pseudouridine or a modified nucleoside, therebyinducing a mammalian cell to produce a recombinant protein.

In another embodiment, an RNA, oligoribonucleotide, orpolyribonucleotide molecule of methods and compositions of the presentinvention is translated in the cell more efficiently than an unmodifiedRNA molecule with the same sequence. In another embodiment, the RNA,oligoribonucleotide, or polyribonucleotide molecule exhibits enhancedability to be translated by a target cell. In another embodiment,translation is enhanced by a factor of 2-fold relative to its unmodifiedcounterpart. In another embodiment, translation is enhanced by a 3-foldfactor. In another embodiment, translation is enhanced by a 5-foldfactor. In another embodiment, translation is enhanced by a 7-foldfactor. In another embodiment, translation is enhanced by a 10-foldfactor. In another embodiment, translation is enhanced by a 15-foldfactor. In another embodiment, translation is enhanced by a 20-foldfactor. In another embodiment, translation is enhanced by a 50-foldfactor. In another embodiment, translation is enhanced by a 100-foldfactor. In another embodiment, translation is enhanced by a 200-foldfactor. In another embodiment, translation is enhanced by a 500-foldfactor. In another embodiment, translation is enhanced by a 1000-foldfactor. In another embodiment, translation is enhanced by a 2000-foldfactor. In another embodiment, the factor is 10-1000-fold. In anotherembodiment, the factor is 10-100-fold. In another embodiment, the factoris 10-200-fold. In another embodiment, the factor is 10-300-fold. Inanother embodiment, the factor is 10-500-fold. In another embodiment,the factor is 20-1000-fold. In another embodiment, the factor is30-1000-fold. In another embodiment, the factor is 50-1000-fold. Inanother embodiment, the factor is 100-1000-fold. In another embodiment,the factor is 200-1000-fold. In another embodiment, translation isenhanced by any other significant amount or range of amounts. Eachpossibility represents a separate embodiment of the present invention.

Methods of determining translation efficiency are well known in the art,and include, e.g. measuring the activity of an encoded reporter protein(e.g. luciferase or renilla [Examples herein] or green fluorescentprotein [Wall A A, Phillips A M et al, Effective translation of thesecond cistron in two Drosophila dicistronic transcripts is determinedby the absence of in-frame AUG codons in the first cistron. J Biol Chem2005; 280(30): 27670-8]), or measuring radioactive label incorporatedinto the translated protein (Ngosuwan J, Wang N M et al, Roles ofcytosolic Hsp70 and Hsp40 molecular chaperones in post-translationaltranslocation of presecretory proteins into the endoplasmic reticulum. JBiol Chem 2003; 278(9): 7034-42). Each method represents a separateembodiment of the present invention.

In some expression studies provided herein, translation was measuredfrom RNA complexed to Lipofectin® (Gibco BRL, Gaithersburg, Md., USA)and injected into the tail vein of mice. In the spleen lysates,pseudouridine-modified RNA was translated significantly more efficientlythan unmodified RNA (FIG. 17B). Under the conditions utilized herein,efficiency of transfection-based methods of the present inventioncorrelates with the ability of the transfection reagent to penetrateinto tissues, providing an explanation for why the effect was mostpronounced in spleen cells. Splenic blood flow is an open system, withblood contents directly contacting red and white pulp elements includinglymphoid cells.

In another experiment, in vitro phosphorylation assays were performedusing recombinant human PKR and its substrate, eIF2α in the presence ofcapped, renilla-encoding mRNA (0.5 and 0.05 ng/μl). mRNA containingpseudouridine (ψ) did not activate PKR, as detected by lack of bothself-phosphorylation of PKR and phosphorylation of eIF2α, while RNAwithout nucleoside modification and mRNA with m5C modification activatedPKR. Phosphorylated eIF2α is known to block initiation of mRNAtranslation, therefore lack of phosphorylation enables, in anotherembodiment, enhanced translation of the mRNA containing pseudouridine(ψ).

In another embodiment, the enhanced translation is in a cell (relativeto translation in the same cell of an unmodified RNA molecule with thesame sequence; Examples 13-14). In another embodiment, the enhancedtranslation is in vitro (e.g. in an in vitro translation mix or areticulocyte lysate; Examples 13-14. In another embodiment, the enhancedtranslation is in vivo (Example 13). In each case, the enhancedtranslation is relative to an unmodified RNA molecule with the samesequence, under the same conditions. Each possibility represents aseparate embodiment of the present invention.

In another embodiment, the RNA, oligoribonucleotide, orpolyribonucleotide molecule of methods and compositions of the presentinvention is significantly less immunogenic than an unmodified invitro-synthesized RNA molecule with the same sequence. In anotherembodiment, the modified RNA molecule is 2-fold less immunogenic thanits unmodified counterpart. In another embodiment, immunogenicity isreduced by a 3-fold factor. In another embodiment, immunogenicity isreduced by a 5-fold factor. In another embodiment, immunogenicity isreduced by a 7-fold factor. In another embodiment, immunogenicity isreduced by a 10-fold factor. In another embodiment, immunogenicity isreduced by a 15-fold factor. In another embodiment, immunogenicity isreduced by a 20-fold factor. In another embodiment, immunogenicity isreduced by a 50-fold factor. In another embodiment, immunogenicity isreduced by a 100-fold factor. In another embodiment, immunogenicity isreduced by a 200-fold factor. In another embodiment, immunogenicity isreduced by a 500-fold factor. In another embodiment, immunogenicity isreduced by a 1000-fold factor. In another embodiment, immunogenicity isreduced by a 2000-fold factor. In another embodiment, immunogenicity isreduced by another fold difference.

In another embodiment, “significantly less immunogenic” refers to adetectable decrease in immunogenicity. In another embodiment, the termrefers to a fold decrease in immunogenicity (e.g. one of the folddecreases enumerated above). In another embodiment, the term refers to adecrease such that an effective amount of the RNA, oligoribonucleotide,or polyribonucleotide molecule can be administered without triggering adetectable immune response. In another embodiment, the term refers to adecrease such that the RNA, oligoribonucleotide, or polyribonucleotidemolecule can be repeatedly administered without eliciting an immuneresponse sufficient to detectably reduce expression of the recombinantprotein. In another embodiment, the decrease is such that the RNA,oligoribonucleotide, or polyribonucleotide molecule can be repeatedlyadministered without eliciting an immune response sufficient toeliminate detectable expression of the recombinant protein.

“Effective amount” of the RNA, oligoribonucleotide, orpolyribonucleotide molecule refers, in another embodiment, to an amountsufficient to exert a therapeutic effect. In another embodiment, theterm refers to an amount sufficient to elicit expression of a detectableamount of the recombinant protein. Each possibility represents aseparate embodiment of the present invention.

Reduced immunogenicity of RNA, oligoribonucleotide, andpolyribonucleotide molecules of the present invention is demonstratedherein (Examples 4-11).

Methods of determining immunogenicity are well known in the art, andinclude, e.g. measuring secretion of cytokines (e.g. IL-12, IFN-α,TNF-α, RANTES, MIP-1α or β, IL-6, IFN-β, or IL-8; Examples herein),measuring expression of DC activation markers (e.g. CD83, HLA-DR, CD80and CD86; Examples herein), or measuring ability to act as an adjuvantfor an adaptive immune response. Each method represents a separateembodiment of the present invention.

In another embodiment, the relative immunogenicity of the modifiednucleotide and its unmodified counterpart are determined by determiningthe quantity of the modified nucleotide required to elicit one of theabove responses to the same degree as a given quantity of the unmodifiednucleotide. For example, if twice as much modified nucleotide isrequired to elicit the same response, than the modified nucleotide istwo-fold less immunogenic than the unmodified nucleotide.

In another embodiment, the relative immunogenicity of the modifiednucleotide and its unmodified counterpart are determined by determiningthe quantity of cytokine (e.g. IL-12, IFN-α, TNF-α, RANTES, MIP-1α or β,IL-6, IFN-β, or IL-8) secreted in response to administration of themodified nucleotide, relative to the same quantity of the unmodifiednucleotide. For example, if one-half as much cytokine is secreted, thanthe modified nucleotide is two-fold less immunogenic than the unmodifiednucleotide. In another embodiment, background levels of stimulation aresubtracted before calculating the immunogenicity in the above methods.Each possibility represents a separate embodiment of the presentinvention.

In another embodiment, a method of present invention further comprisesmixing the RNA, oligoribonucleotide, or polyribonucleotide molecule witha transfection reagent prior to the step of contacting. In anotherembodiment, a method of present invention further comprisesadministering the RNA, oligoribonucleotide, or polyribonucleotidemolecule together with the transfection reagent. In another embodiment,the transfection reagent is a cationic lipid reagent (Example 6).

In another embodiment, the transfection reagent is a lipid-basedtransfection reagent. In another embodiment, the transfection reagent isa protein-based transfection reagent. In another embodiment, thetransfection reagent is a polyethyleneimine based transfection reagent.In another embodiment, the transfection reagent is calcium phosphate. Inanother embodiment, the transfection reagent is Lipofectin® orLipofectamine®. In another embodiment, the transfection reagent is anyother transfection reagent known in the art.

In another embodiment, the transfection reagent forms a liposome.Liposomes, in another embodiment, increase intracellular stability,increase uptake efficiency and improve biological activity. In anotherembodiment, liposomes are hollow spherical vesicles composed of lipidsarranged in a similar fashion as those lipids which make up the cellmembrane. They have, in another embodiment, an internal aqueous spacefor entrapping water soluble compounds and range in size from 0.05 toseveral microns in diameter. In another embodiment, liposomes candeliver RNA to cells in a biologically active form.

Each type of transfection reagent represents a separate embodiment ofthe present invention.

In another embodiment, the target cell of methods of the presentinvention is an antigen—presenting cell. In another embodiment, the cellis an animal cell. In another embodiment, the cell is a dendritic cell(Example 14). In another embodiment, the cell is a neural cell. Inanother embodiment, the cell is a brain cell (Example 16). In anotherembodiment, the cell is a spleen cell. In another embodiment, the cellis a lymphoid cell. In another embodiment, the cell is a lung cell(Example 16). In another embodiment, the cell is a skin cell. In anotherembodiment, the cell is a keratinocyte. In another embodiment, the cellis an endothelial cell. In another embodiment, the cell is an astrocyte,a microglial cell, or a neuron (Example 16). In another embodiment, thecell is an alveolar cell (Example 16). In another embodiment, the cellis a surface alveolar cell (Example 16). In another embodiment, the cellis an alveolar macrophage. In another embodiment, the cell is analveolar pneumocyte. In another embodiment, the cell is a vascularendothelial cell. In another embodiment, the cell is a mesenchymal cell.In another embodiment, the cell is an epithelial cell. In anotherembodiment, the cell is a hematopoietic cell. In another embodiment, thecell is colonic epithelium cell. In another embodiment, the cell is alung epithelium cell. In another embodiment, the cell is a bone marrowcell.

In other embodiments, the target cell is a Claudius' cell, Hensen cell,Merkel cell, Muller cell, Paneth cell, Purkinje cell, Schwann cell,Sertoli cell, acidophil cell, acinar cell, adipoblast, adipocyte, brownor white alpha cell, amacrine cell, beta cell, capsular cell,cementocyte, chief cell, chondroblast, chondrocyte, chromaffin cell,chromophobic cell, corticotroph, delta cell, Langerhans cell, folliculardendritic cell, enterochromaffin cell, ependymocyte, epithelial cell,basal cell, squamous cell, endothelial cell, transitional cell,erythroblast, erythrocyte, fibroblast, fibrocyte, follicular cell, germcell, gamete, ovum, spermatozoon, oocyte, primary oocyte, secondaryoocyte, spermatid, spermatocyte, primary spermatocyte, secondaryspermatocyte, germinal epithelium, giant cell, glial cell, astroblast,astrocyte, oligodendroblast, oligodendrocyte, glioblast, goblet cell,gonadotroph, granulosa cell, haemocytoblast, hair cell, hepatoblast,hepatocyte, hyalocyte, interstitial cell,’ juxtaglomerular cell,keratinocyte, keratocyte, lemmal cell, leukocyte, granulocyte, basophil,eosinophil, neutrophil, lymphoblast, B-lymphoblast, T-lymphoblast,lymphocyte, B-lymphocyte, T-lymphocyte, helper induced T-lymphocyte, Th1T-lymphocyte, Th2 T-lymphocyte, natural killer cell, thymocyte,macrophage, Kupffer cell, alveolarmacrophage, foam cell, histiocyte,luteal cell, lymphocytic stem cell, lymphoid cell, lymphoid stem cell,macroglial cell, mammotroph, mast cell, medulloblast, megakaryoblast,megakaryocyte, melanoblast, melanocyte, mesangial cell, mesothelialcell, metamyelocyte, monoblast, monocyte, mucous neck cell, muscle cell,cardiac muscle cell, skeletal muscle cell, smooth muscle cell,myelocyte, myeloid cell, myeloid stem cell, myoblast, myoepithelialcell, myofibrobast, neuroblast, neuroepithelial cell, neuron,odontoblast, osteoblast, osteoclast, osteocyte, oxyntic cell,parafollicular cell, paraluteal cell, peptic cell, pericyte, peripheralblood mononuclear cell, phaeochromocyte, phalangeal cell, pinealocyte,pituicyte, plasma cell, platelet, podocyte, proerythroblast,promonocyte, promyeloblast, promyelocyte, pronormoblast, reticulocyte,retinal pigment epithelial cell, retinoblast, small cell, somatotroph,stem cell, sustentacular cell, teloglial cell, or zymogenic cell. Eachpossibility represents a separate embodiment of the present invention.

A variety of disorders may be treated by employing methods of thepresent invention including, inter alia, monogenic disorders, infectiousdiseases, acquired disorders, cancer, and the like. Exemplary monogenicdisorders include ADA deficiency, cystic fibrosis,familial-hypercholesterolemia, hemophilia, chronic ganulomatous disease,Duchenne muscular dystrophy, Fanconi anemia, sickle-cell anemia,Gaucher's disease, Hunter syndrome, X-linked SCID, and the like. Inanother embodiment, the disorder treated involves one of the proteinslisted below. Each possibility represents a separate embodiment of thepresent invention.

In another embodiment, the recombinant protein encoded by an RNA,oligoribonucleotide, or polyribonucleotide molecule of methods andcompositions of the present invention is ecto-nucleoside triphosphatediphosphohydrolase.

In another embodiment, the recombinant protein is erythropoietin (EPO).In other embodiments, the encoded recombinant protein is ABCA4; ABCD3;ACADM; AGL; AGT; ALDH4AI; ALPL; AMPD1; APOA2; AVSD1; BRCD2; C1QA; C1QB;C1QG; CBA; C8B; CACNA1S; CCV; CD3Z; CDC2L1; CHML; CHS1; CIAS1; CLCNKB;CMD1A; CMH2; CMM; COL11A1; COL8A2; COL9A2; CPT2; CRB1; CSE; CSF3R; CTPA;CTSK; DBT; DIO1; DISC1; DPYD; EKV; ENO1; ENO1P; EPB41; EPHX1; F13B; F5;FCGR2A; FCGR2B; FCGR3A; FCHL; FH; FMO3; FMO4; FUCA1; FY; GALE; GBA;GFND; GJA8; GJB3; GLC3B; HF1; HMGCL; HPC1; HRD; HRPT2; HSD3B2; HSPG2;KCNQ4; KCS; KIF1B; LAMB3; LAMC2; LGMD1B; LMNA; LOR; MCKD1; MCL1; MPZ;MTHFR; MTR; MUTYH; MYOC; NB; NCF2; NEM1; NPHS2; NPPA; NRAS; NTRK1;OPTA2; PBX1; PCHC; PGD; PHA2A; PHGDH; PKLR; PKP1; PLA2G2A; PLOD; PPDX;PPT1; PRCC; PRG4; PSEN2; PTOS1; REN; RFX5; RHD; RMD1; RPE65; SCCD;SERPINC1; SJS1; SLC19A2; SLC2A1; SPG23; SPTA1; TAL1; TNFSF6; TNNT2;TPM3; TSHB; UMPK; UOX; UROD; USH2A; VMGLOM; VWS; WS2B; ABCB11; ABCG5;ABCG8; ACADL; ACP1; AGXT; AHHR; ALMS1; ALPP; ALS2; APOB; BDE; BDMR; BJS;BMPR2; CHRNA1; CMCWTD; CNGA3; COL3A1; COL4A3; COL4A4; COL6A3; CPSI;CRYGA; CRYGEP1; CYP1B1; CYP27A1; DBI; DES; DYSF; EDAR; EFEMPI; EIF2AK3;ERCC3; FSHR; GINGF; GLC1B; GPD2; GYPC; HADHA; HADHB; HOXD13; HPE2; IGKC;IHH; IRSI; ITGA6; KHK; KYNU; LCT; LHCGR; LSFC; MSH2; MSH6; NEB; NMTC;NPHP1; PAFAH1P1; PAX3; PAX8; PMS1; PNKD; PPH1; PROC; REG1A; SAG; SFTPB;SLC11A1; SLC3A1; SOS1; SPG4; SRD5A2; TCL4; TGFA; TMD; TPO; UGT1A@; UV24;WSS; XDH; ZAP70; ZFHX1B; ACAA1; AGS1; AGTR1; AHSG; AMT; ARMET; BBS3;BCHE; BCPM; BTD; CASR; CCR2; CCR5; CDL1; CMT2B; COL7A1; CP; CPO; CRV;CTNNB1; DEM; ETM1; FANCD2; FIH; FOXL2; GBE1; GLB1; GLC1C; GNAI2; GNATI;GP9; GPX1; HGD; HRG; ITIH1; KNG; LPP; LRS1; MCCCI; MDS1; MHS4; MITF;MLH1; MYL3; MYMY; OPA1; P2RY12; PBXP1; PCCB; POU1F1; PPARG; PROS1;PTHR1; RCA1; RHO; SCAT; SCLC1; SCN5A; SI; SLC25A20; SLC2A2; TF; TGFBR2;THPO; THRB; TKT; TM4SF1; TRH; UMPS; UQCRC1; USH3A; VHL; WS2A; XPC;ZNF35; ADH1B; ADH1C; AFP; AGA; AIH2; ALB; ASMD; BFHD; CNGA1; CRBM; DCK;DSPP; DTDP2; ELONG; ENAM; ETFDH; EVC; F11; FABP2; FGA; FGB; FGFR3; FGG;FSHMD1A; GC; GNPTA; GNRHR; GYPA; HCA; HCL2; HD; HTN3; HVBS6; IDUA; IF;JPD; KIT; KLKB1; LQT4; MANBA; MLLT2; MSX1; MTP; NR3C2; PBT; PDE6B; PEE1;PITX2; PKD2; QDPR; SGCB; SLC25A4; SNCA; SOD3; STATH; TAPVR1; TYS; WBS2;WFS1; WHCR; ADAMTS2; ADRB2; AMCN; AP3BI; APC; ARSB; B4GALT7; BHR1; C6;C7; CCAL2; CKN1; CMDJ; CRHBP; CSF1R; DHFR; DIAPH1; DTR; EOS; EPD; ERVR;F12; FBN2; GDNF; GHR; GLRA1; GM2A; HEXB; HSD17B4; ITGA2; KFS; LGMD1A;LOX; LTC4S; MAN2A1; MCC; MCCC2; MSH3; MSX2; NR3C1; PCSK1; PDE6A; PFBI;RASAI; SCZDI; SDHA; SGCD; SLC22A5; SLC26A2; SLC6A3; SM1; SMA@; SMN1;SMN2; SPINK5; TCOF1; TELAB1; TGFBI; ALDH5A1; ARG1; AS; ASSP2; BCKDHB;BF; C2; C4A; CDKN1A; COL10A1; COL11A2; CYP21A2; DYX2; EJM1; ELOVL4;EPM2A; ESR1; EYA4; F13A1; FANCE; GCLC; GJA1; GLYS1; GMPR; GSE; HCR; HFE;HLA-A; HLA-DPBI; HLA-DRA; HPFH; ICS1; IDDM1; IFNGR1; IGAD1; IGF2R; ISCW;LAMA2; LAP; LCA5; LPA; MCDR1; MOCS1; MUT; MYB; NEU1; NKS1; NYS2; OA3;ODDD; OFC1; PARK2; PBCA; PBCRA1; PDB1; PEX3; PEX6; PEX7; PKHD1; PLA2G7;PLG; POLH; PPAC; PSORS1; PUJO; RCD1; RDS; RHAG; RP14; RUNX2; RWS; SCA1;SCZD3; SIASD; SOD2; ST8; TAPl; TAP2; TFAP2B; TNDM; TNF; TPBG; TPMT;TULP1; WISP3; AASS; ABCB1; ABCB4; ACHE; AQP1; ASL; ASNS; AUTS1; BPGM;BRAF; C7orf2; CACNA2D1; CCM1; CD36; CFTR; CHORDOMA; CLCN1; CMH6; CMT2D;COL1A2; CRS; CYMD; DFNA5; DLD; DYT11; EEC1; ELN; ETV1; FKBP6; GCK;GHRHR; GHS; GLI3; GPDS1; GUSB; HLXB9; HOXA13; HPFH2; HRX; IAB; IMMP2L;KCNH2; LAMB1; LEP; MET; NCF1; NM; OGDH; OPN1SW; PEX1; PGAM2; PMS2; PON1;PPP1R3A; PRSS1; PTC; PTPN12; RP10; RP9; SERPINE1; SGCE; SHFM1; SHH;SLC26A3; SLC26A4; SLOS; SMAD1; TBXAS1; TWIST; ZWS1; ACHM3; ADRB3; ANK1;CA1; CA2; CCAL1; CLN8; CMT4A; CNGB3; COH1; CPP; CRH; CYP11B1; CYP11B2;DECR1; DPYS; DURS1; EBS1; ECA1; EGI; EXT1; EYA1; FGFR1; GNRH1; GSR;GULOP; HR; KCNQ3; KFM; KWE; LGCR; LPL; MCPH1; MOS; MYC; NAT1; NAT2;NBS1; PLAT; PLEC1; PRKDC; PXMP3; RP1; SCZD6; SFIPC; SGM1; SPG5A; STAR;TG; TRPS1; TTPA; VMD1; WRN; ABCA1; ABL1; ABO; ADAMTS13; AK1; ALAD;ALDH1A1; ALDOB; AMBP; AMCD1; ASS; BDMF; BSCL; C5; CDKN2A; CHAC; CLA1;CMD1B; COL5A1; CRAT; DBH; DNAI1; DYS; DYT1; ENG; FANCC; FBP1; FCMD;FRDA; GALT; GLDC; GNE; GSM1; GSN; HSD17B3; HSN1; IBM2; INVS; JBTS1;LALL; LCCS1; LCCS; LGMD2H; LMX1B; MLLT3; MROS; MSSE; NOTCH1; ORM1;PAPPA; PIP5K1B; PTCH; PTGS1; RLN1; RLN2; RMRP; ROR2; RPD1; SARDH;SPTLC1; STOM; TDFA; TEK; TMC1; TRIM32; TSC1; TYRP1; XPA; CACNB2;COL17A1; CUBN; CXCL12; CYP17; CYP2C19; CYP2C9; EGR2; EMX2; ERCC6; FGFR2;HK1; HPS1; IL2RA; LGI1; LIPA; MAT1A; MBL2; MKI67; MXI1; NODAL; OAT;OATL3; PAX2; PCBD; PEO1; PHYH; PNLIP; PSAP; PTEN; RBP4; RDPA; RET;SFTPA1; SFTPD; SHFM3; SIAL; THC2; TLX1; TNFRSF6; UFS; UROS; AA; ABCC8;ACAT1; ALX4; AMPD3; ANC; APOA1; APOA4; APOC3; ATM; BSCL2; BWS; CALCA;CAT; CCND1; CD3E; CD3G; CD59; CDKN1C; CLN2; CNTF; CPT1A; CTSC; DDB1;DDB2; DHCR7; DLAT; DRD4; ECB2; ED4; EVR1; EXT2; F2; FSHB; FTH1; G6PT1;G6PT2; GIF; HBB; HBBP1; HBD; HBE1; HBG1; HBG2; HMBS; HND; HOMG2; HRAS;HVBS1; IDDM2; IGER; INS; JBS; KCNJ11; KCNJ1; KCNQ1; LDHA; LRP5; MEN1;MILL; MYBPC3; MYO7A; NNO1; OPPG; OPTB1; PAX6; PC; PDX1; PGL2; PGR; PORC;PTH; PTS; PVRL1; PYGM; RAG1; RAG2; ROM1; RRAS2; SAA1; SCA5; SCZD2; SDHD;SERPING1; SMPD1; TCIRG1; TCL2; TECTA; TH; TREH; TSG101; TYR; USH1C;VMD2; VRNI; WT1; WT2; ZNF145; A2M; AAAS; ACADS; ACLS; ACVRL1; ALDH2;AMHR2; AOM; AQP2; ATD; ATP2A2; BDC; C1R; CD4; CDK4; CNA1; COL2A1;CYP27B1; DRPLA; ENUR2; FEOM1; FGF23; FPF; GNB3; GNS; HAL; HBP1; HMGA2;HMN2; HPD; IGF1; KCNA1; KERA; KRAS2; KRT1; KRT2A; KRT3; KRT4; KRT5;KRT6A; KRT6B; KRTHB6; LDHB; LYZ; MGCT; MPE; MVK; MYL2; OAP; PAH; PPKB;PRB3; PTPN11; PXR1; RLS; RSN; SAS; SAX1; SCA2; SCNN1A; SMAL; SPPM;SPSMA; TBX3; TBX5; TCF1; TPI1; TSC3; ULR; VDR; VWF; ATP7B; BRCA2; BRCD1;CLN5; CPB2; ED2; EDNRB; ENUR1; ERCC5; F10; F7; GJB2; GJB6; IPF1; MBS1;MCOR; NYS4; PCCA; RB1; RHOK; SCZD7; SGCG; SLC10A2; SLC25A15; STARP1;ZNF198; ACHM1; ARVD1; BCH; CTAA1; DAD1; DFNB5; EML1; GALC; GCH1; IBGC1;IGH@; IGHC group; IGHG1; IGHM; IGHR; IV; LTBP2; MCOP; MJD; MNG1; MPD1;MPS3C; MYH6; MYH7; NP; NPC2; PABPN1; PSEN1; PYGL; RPGRIP1; SERPINA1;SERPINA3; SERPINA6; SLC7A7; SPG3A; SPTB; TCL1A; TGMI; TITF1; TMIP; TRA@;TSHR; USH1A; VP; ACCPN; AHO2; ANCR; B2M; BBS4; BLM; CAPN3; CDAN1; CDAN3;CLN6; CMH3; CYP19; CYP1A1; CYP1A2; DYX1; EPB42; ETFA; EYCL3; FAH; FBN1;FES; HCVS; HEXA; IVD; LCS1; LIPC; MY05A; OCA2; OTSC1; PWCR; RLBP1;SLC12A1; SPG6; TPM1; UBE3A; WMS; ABCC6; ALDOA; APRT; ATP2A1; BBS2;CARD15; CATM; CDH1; CETP; CHST6; CLN3; CREBBP; CTH; CTM; CYBA; CYLD;DHS; DNASE1; DPEP1; ERCC4; FANCA; GALNS; GAN; HAGH; HBA1; HBA2; HBHR;HBQ1; HBZ; HBZP; HP; HSD11B2; IL4R; LIPB; MC1R; MEFV; METC2TA; MLYCD;MMVP1; PHKB; PHKG2; PKD1; PKDTS; PMM2; PXE; SALL1; SCA4; SCNN1B; SCNN1G;SLC12A3; TAT; TSC2; VDI; WT3; ABR; ACACA; ACADVL; ACE; ALDH3A2; APOH;ASPA; AXIN2; BCL5; BHD; BLMH; BRCA1; CACD; CCA1; CCZS; CHRNB1; CHRNE;CMT1A; COL1A1; CORD5; CTNS; EPX; ERBB2; G6PC; GAA; GALK1; GCGR; GFAP;GH1; GH2; GP1BA; GPSC; GUCY2D; ITGA2B; ITGB3; ITGB4; KRT10; KRT12;KRT13; KRT14; KRT14L1; KRT14L2; KRT14L3; KRT16; KRT16L1; KRT16L2; KRT17;KRT9; MAPT; MDB; MDCR; MGI; MHS2; MKS1; MPO; MYO15A; NAGLU; NAPB; NF1;NME1; P4HB; PAFAH1B1; PECAM1; PEX12; PHB; PMP22; PRKAR1A; PRKCA;PRKWNK4; PRP8; PRPF8; PTLAH; RARA; RCV1; RMSA1; RP17; RSS; SCN4A;SERPINF2; SGCA; SGSH; SHBG; SLC2A4; SLC4A1; SLC6A4; SMCR; SOST; SOX9;SSTR2; SYM1; SYNSI; TCF2; THRA; TIMP2; TOC; TOP2A; TP53; TRIM37; VBCH;ATP8B1; BCL2; CNSN; CORD1I; CYB5; DCC; F5F8D; FECH; FEO; LAMA3; LCFS2;MADH4; MAFD1; MC2R; MCL; MYP2; NPC1; SPPK; TGFBRE; TGIF; TTR; AD2; AMH;APOC2; APOE; ATHS; BAX; BCKDHA; BCL3; BFIC; C3; CACNA1A; CCO; CEACAM5;COMP; CRX; DBA; DDU; DFNA4; DLL3; DM1; DMWD; E11S; ELA2; EPOR; ERCC2;ETFB; EXT3; EYCLI; FTL; FUT1; FUT2; FUT6; GAMT; GCDH; GPI; GUSM; HB1;HCL1; HHC2; HHC3; ICAM3; INSR; JAK3; KLK3; LDLR; LHB; LIG1; LOH19CR1;LYL1; MAN2B1; MCOLN1; MDRV; MLLT1; NOTCH3; NPHS1; OFC3; OPA3; PEPD;PRPF31; PRTN3; PRX; PSG1; PVR; RYR1; SLC5A5; SLC7A9; STK11; TBXA2R;TGFB1; TNNI3; TYROBP; ADA; AHCY; AVP; CDAN2; CDPD1; CHED1; CHED2;CHRNA4; CST3; EDN3; EEGV1; FTLL1; GDF5; GNAS; GSS; HNF4A; JAG1; KCNQ2;MKKS; NBIA1; PCK1; PI3; PPCD; PPGB; PRNP; THBD; TOP1; AIRE; APP; CBS;COL6A1; COL6A2; CSTB; DCR; DSCR1; FPDMM; HLCS; HPE1; ITGB2; KCNE1; KNO;PRSS7; RUNX1; SOD1; TAM; ADSL; ARSA; BCR; CECR; CHEK2; COMT; CRYBB2;CSF2RB; CTHM; CYP2D6; CYP2D7P1; DGCR; DIA1; EWSR1; GGT1; MGCR; MN1;NAGA; NE2; OGS2; PDGFB; PPARA; PRODH; SCO2; SCZD4; SERPIND1; SLC5AI;SOXI0; TCN2; TIMP3; TST; VCF; ABCD1; ACTL1; ADFN; AGMX2; AHDS; AIC;AIED; AIH3; ALAS2; AMCD; AMELX; ANOP1; AR; ARAF1; ARSC2; ARSE; ARTS;ARX; ASAT; ASSP5; ATP7A; ATRX; AVPR2; BFLS; BGN; BTK; BZX; C1HR;CACNA1F; CALB3; CBBM; CCT; CDR1; CFNS; CGF1; CHM; CHR39C; CIDX; CLA2;CLCN5; CLS; CMTX2; CMTX3; CND; COD1; COD2; COL4A5; COL4A6; CPX; CVD1;CYBB; DCX; DFN2; DFN4; DFN6; DHOF; DIAPH2; DKC1; DMD; DSS; DYT3; EBM;EBP; ED1; ELK1; EMD; EVR2; F8; F9; FCP1; FDPSL5; FGD1; FGS1; FMR1; FMR2;G6PD; GABRA3; GATA1; GDI1; GDXY; GJB1; GK; GLA; GPC3; GRPR; GTD; GUST;HMS1; HPRT1; HPT; HTC2; HTR2C; HYR; IDS; IHG1; IL2RG; INDX; IP1; IP2;JMS; KAL1; KFSD; LICAM; LAMP2; MAA; MAFD2; MAOA; MAOB; MCF2; MCS; MEAX;MECP2; MF4; MGCI; MIC5; MID1; MLLT7; MILS; MRSD; MRX14; MRX1; MRX20;MRX2; MRX3; MRX40; MRXA; MSD; MTMI; MYCL2; MYPI; NDP; NHS; NPHLI; NROBI;NSX; NYSI; NYX; OAI; OASD; OCRL; ODTI; OFD1; OPA2; OPD1; OPEM; OPN1LW;OPN1MW; OTC; P3; PDHA1; PDR; PFC; PFKFB1; PGK1; PGK1P1; PGS; PHEX;PHKA1; PHKA2; PHP; PIGA; PLP1; POF1; POLA; POU3F4; PPMX; PRD; PRPSI;PRPS2; PRS; RCCP2; RENBP; RENS1; RP2; RP6; RPGR; RPS4X; RPS6KA3; RS1;S11; SDYS; SEDL; SERPINA7; SH2D1A; SHFM2; SLC25A5; SMAX2; SRPX; SRS;STS; SYN1; SYP; TAF1; TAZ; TBX22; TDD; TFE3; THAS; THC; TIMM8A; TIMP1;TKCR; TNFSF5; UBE1; UBE2A; WAS; WSN; WTS; WWS; XIC; XIST; XK; XM; XS;ZFX; ZIC3; ZNF261; ZNF41; ZNF6; AMELY; ASSP6; AZFI; AZF2; DAZ; GCY;RPS4Y; SMCY; SRY; ZFY; ABAT; AEZ; AFA; AFD1; ASAH1; ASD1; ASMT; CCAT;CECR9; CEPA; CLA3; CLN4; CSF2RA; CTSI; DF; DIH1; DWS; DYT2; DYT4; EBR3;ECT; EEF1A1L14; EYCL2; FANCB; GCSH; GCSL; GIP; GTS; HHG; HMI; HOAC;HOKPP2; HRPT1; HSD3B3; HTC1; HV1S; ICHQ; ICR1; ICR5; IL3RA; KAL2; KMS;KRT18; KSS; LCAT; LHON; LIMM; MANBB; MCPH2; MEB; MELAS; MIC2; MPFD; MS;MSS; MTATP6; MTCOI; MTCO3; MTCYB; MTND1; MTND2; MTND4; MTND5; MTND6;MTRNR1; MTRNR2; MTTE; MTTG; MTTI; MTTK; MTTL1; MTTL2; MTTN; MTTP; MTTS1;NAMSD; OCD1; OPD2; PCK2; PCLD; PCOS1; PFKM; PKD3; PRCA1; PRO1; PROP1;RBS; RFXAP; RP; SHOX; SLC25A6; SPG5B; STO; SUOX; THM; or TTD. Eachrecombinant protein represents a separate embodiment of the presentinvention.

In another embodiment, the present invention provides a method fortreating anemia in a subject, comprising contacting a cell of thesubject with an in vitro-synthesized RNA molecule, the in vitrosynthesized RNA molecule encoding erythropoietin, thereby treatinganemia in a subject. In another embodiment, the in vitro-synthesized RNAmolecule further comprises a pseudouridine or a modified nucleoside.Each possibility represents a separate embodiment of the presentinvention. In another embodiment, the cell is a subcutaneous tissuecell. In another embodiment, the cell is a lung cell. In anotherembodiment, the cell is a fibroblast. In another embodiment, the cell isa lymphocyte. In another embodiment, the cell is a smooth muscle cell.In another embodiment, the cell is any other type of cell known in theart. Each possibility represents a separate embodiment of the presentinvention.

In another embodiment, the present invention provides a method fortreating a vasospasm in a subject, comprising contacting a cell of thesubject with an in vitro-synthesized RNA molecule, the invitro-synthesized RNA molecule encoding inducible nitric oxide synthase(iNOS), thereby treating a vasospasm in a subject.

In another embodiment, the present invention provides a method forimproving a survival rate of a cell in a subject, comprising contactingthe cell with an in vitro-synthesized RNA molecule, the invitro-synthesized RNA molecule encoding a heat shock protein, therebyimproving a survival rate of a cell in a subject.

In another embodiment, the cell whose survival rate is improved is anischemic cell. In another embodiment, the cell is not ischemic. Inanother embodiment, the cell has been exposed to an ischemicenvironment. In another embodiment, the cell has been exposed to anenvironmental stress. Each possibility represents a separate embodimentof the present invention.

In another embodiment, the present invention provides a method fordecreasing an incidence of a restenosis of a blood vessel following aprocedure that enlarges the blood vessel, comprising contacting a cellof the blood vessel with an in vitro-synthesized RNA molecule, the invitro-synthesized RNA molecule encoding a heat shock protein, therebydecreasing an incidence of a restenosis in a subject.

In another embodiment, the procedure is an angioplasty. In anotherembodiment, the procedure is any other procedure known in the art thatenlarges the blood vessel. Each possibility represents a separateembodiment of the present invention.

In another embodiment, the present invention provides a method forincreasing a hair growth from a hair follicle is a scalp of a subject,comprising contacting a cell of the scalp with an in vitro-synthesizedRNA molecule, the in vitro-synthesized RNA molecule encoding atelomerase or an immunosuppressive protein, thereby increasing a hairgrowth from a hair follicle.

In another embodiment, the immunosuppressive protein isα-melanocyte-stimulating hormone (α-MSH). In another embodiment, theimmunosuppressive protein is transforming growth factor-β 1 (TGF-β1). Inanother embodiment, the immunosuppressive protein is insulin-like growthfactor-I (IGF-I). In another embodiment, the immunosuppressive proteinis any other immunosuppressive protein known in the art. Eachpossibility represents a separate embodiment of the present invention.

In another embodiment, the present invention provides a method ofinducing expression of an enzyme with antioxidant activity in a cell,comprising contacting the cell with an in vitro-synthesized RNAmolecule, the in vitro-synthesized RNA molecule encoding the enzyme,thereby inducing expression of an enzyme with antioxidant activity in acell.

In one embodiment, the enzyme is catalase. In another embodiment, theenzyme is glutathione peroxidase. In another embodiment, the enzyme isphospholipid hydroperoxide glutathione peroxidase. In anotherembodiment, the enzyme is superoxide dismutase-1. In another embodiment,the enzyme is superoxide dismutase-2. In another embodiment, the enzymeis any other enzyme with antioxidant activity that is known in the art.Each possibility represents a separate embodiment of the presentinvention.

In another embodiment, the present invention provides a method fortreating cystic fibrosis in a subject, comprising contacting a cell ofthe subject with an in vitro-synthesized RNA molecule, the invitro-synthesized RNA molecule encoding Cystic Fibrosis TransmembraneConductance Regulator (CFTR), thereby treating cystic fibrosis in asubject.

In another embodiment, the present invention provides a method fortreating an X-linked agammaglobulinemia in a subject, comprisingcontacting a cell of the subject with an in vitro synthesized RNAmolecule, the in vitro-synthesized RNA molecule encoding a Bruton'styrosine kinase, thereby treating an X-linked agammaglobulinemia.

In another embodiment, the present invention provides a method fortreating an adenosine deaminase severe combined immunodeficiency (ADASCID) in a subject, comprising contacting a cell of the subject with anin vitro-synthesized RNA molecule, the in vitro-synthesized RNA moleculeencoding an ADA, thereby treating an ADA SCID.

In another embodiment, the present invention provides a method forreducing immune responsiveness of the skin and improving skin pathology,comprising contacting a cell of the subject with an in vitro-synthesizedRNA molecule, the in vitro-synthesized RNA molecule encoding anecto-nucleoside triphosphate diphosphohydrolase, thereby reducing immuneresponsiveness of the skin and improving skin pathology.

In another embodiment, an RNA molecule or ribonucleotide molecule of thepresent invention is encapsulated in a nanoparticle. Methods fornanoparticle packaging are well known in the art, and are described, forexample, in Bose S, et al (Role of Nucleolin in Human ParainfluenzaVirus Type 3 Infection of Human Lung Epithelial Cells. J. Virol.78:8146. 2004); Dong Y et al.Poly(d,l-lactide-co-glycolide)/montmorillonite nanoparticles for oraldelivery of anticancer drugs. Biomaterials 26:6068. 2005); Lobenberg R.et al (Improved body distribution of 14C-labelled AZT bound tonanoparticles in rats determined by radioluminography. J Drug Target5:171. 1998); Sakuma S R et al (Mucoadhesion of polystyrenenanoparticles having surface hydrophilic polymeric chains in thegastrointestinal tract. Int J Pharm 177:161. 1999); Virovic L et al.Novel delivery methods for treatment of viral hepatitis: an update.Expert Opin Drug Deliv 2:707.2005); and Zimmermann E et al.,Electrolyte- and pH-stabilities of aqueous solid lipid nanoparticle(SLN) dispersions in artificial gastrointestinal media. Eur J PharmBiopharm 52:203.2001). Each method represents a separate embodiment ofthe present invention.

Various embodiments of dosage ranges of compounds of the presentinvention can be used in methods of the present invention. In oneembodiment, the dosage is in the range of 1-10 μg/day. In anotherembodiment, the dosage is 2-10 μg/day. In another embodiment, the dosageis 3-10 μg/day. In another embodiment, the dosage is 5-10 μg/day. Inanother embodiment, the dosage is 2-20 μg/day. In another embodiment,the dosage is 3-20 μg/day. In another embodiment, the dosage is 5-20μg/day. In another embodiment, the dosage is 10-20 μg/day. In anotherembodiment, the dosage is 3-40 μg/day. In another embodiment, the dosageis 5-40 μg/day. In another embodiment, the dosage is 10-40 μg/day. Inanother embodiment, the dosage is 20-40 μg/day. In another embodiment,the dosage is 5-50 μg/day. In another embodiment, the dosage is 10-50μg/day. In another embodiment, the dosage is 20-50 μg/day. In oneembodiment, the dosage is 1-100 μg/day. In another embodiment, thedosage is 2-100 μg/day. In another embodiment, the dosage is 3-100μg/day. In another embodiment, the dosage is 5-100 μg/day. In anotherembodiment the dosage is 10-100 μg/day. In another embodiment the dosageis 20-100 μg/day. In another embodiment the dosage is 40-100 μg/day. Inanother embodiment the dosage is 60-100 μg/day.

In another embodiment, the dosage is 0.1 μg/day. In another embodiment,the dosage is 0.2 μg/day. In another embodiment, the dosage is 0.3μg/day. In another embodiment, the dosage is 0.5 μg/day. In anotherembodiment, the dosage is 1 μg/day. In another embodiment, the dosage is2 mg/day. In another embodiment, the dosage is 3 μg/day. In anotherembodiment, the dosage is 5 μg/day. In another embodiment, the dosage is10 μg/day. In another embodiment, the dosage is 15 μg/day. In anotherembodiment, the dosage is 20 μg/day. In another embodiment, the dosageis 30 μg/day. In another embodiment, the dosage is 40 μg/day. In anotherembodiment, the dosage is 60 μg/day. In another embodiment, the dosageis 80 μg/day. In another embodiment, the dosage is 100 μg/day.

In another embodiment, the dosage is 10 μg/dose. In another embodiment,the dosage is 20 μg/dose. In another embodiment, the dosage is 30μg/dose. In another embodiment, the dosage is 40 μg/dose. In anotherembodiment, the dosage is 60 μg/dose. In another embodiment, the dosageis 80 μg/dose. In another embodiment, the dosage is 100 μg/dose. Inanother embodiment, the dosage is 150 μg/dose. In another embodiment,the dosage is 200 μg/dose. In another embodiment, the dosage is 300μg/dose. In another embodiment, the dosage is 400 μg/dose. In anotherembodiment, the dosage is 600 μg/dose. In another embodiment, the dosageis 800 μg/dose. In another embodiment, the dosage is 1000 μg/dose. Inanother embodiment, the dosage is 1.5 mg/dose. In another embodiment,the dosage is 2 mg/dose. In another embodiment, the dosage is 3 mg/dose.In another embodiment, the dosage is 5 mg/dose. In another embodiment,the dosage is 10 mg/dose. In another embodiment, the dosage is 15mg/dose. In another embodiment, the dosage is 20 mg/dose. In anotherembodiment, the dosage is 30 mg/dose. In another embodiment, the dosageis 50 mg/dose. In another embodiment, the dosage is 80 mg/dose. Inanother embodiment, the dosage is 100 mg/dose.

In another embodiment, the dosage is 10-20 μg/dose. In anotherembodiment, the dosage is 20-30 μg/dose. In another embodiment, thedosage is 20-40 μg/dose. In another embodiment, the dosage is 30-60μg/dose. In another embodiment, the dosage is 40-80 μg/dose. In anotherembodiment, the dosage is 50-100 μg/dose. In another embodiment, thedosage is 50-150 μg/dose. In another embodiment, the dosage is 100-200μg/dose. In another embodiment, the dosage is 200-300 μg/dose. Inanother embodiment, the dosage is 300-400 μg/dose. In anotherembodiment, the dosage is 400-600 μg/dose. In another embodiment, thedosage is 500-800 μg/dose. In another embodiment, the dosage is 800-1000μg/dose. In another embodiment, the dosage is 1000-1500 μg/dose. Inanother embodiment, the dosage is 1500-2000 μg/dose. In anotherembodiment, the dosage is 2-3 mg/dose. In another embodiment, the dosageis 2-5 mg/dose. In another embodiment, the dosage is 2-10 mg/dose. Inanother embodiment, the dosage is 2-20 mg/dose. In another embodiment,the dosage is 2-30 mg/dose. In another embodiment, the dosage is 2-50mg/dose. In another embodiment, the dosage is 2-80 mg/dose. In anotherembodiment, the dosage is 2-100 mg/dose. In another embodiment, thedosage is 3-10 mg/dose. In another embodiment, the dosage is 3-20mg/dose. In another embodiment, the dosage is 3-30 mg/dose. In anotherembodiment, the dosage is 3-50 mg/dose. In another embodiment, thedosage is 3-80 mg/dose. In another embodiment, the dosage is 3-100mg/dose. In another embodiment, the dosage is 5-10 mg/dose. In anotherembodiment, the dosage is 5-20 mg/dose. In another embodiment, thedosage is 5-30 mg/dose. In another embodiment, the dosage is 5-50mg/dose. In another embodiment, the dosage is 5-80 mg/dose. In anotherembodiment, the dosage is 5-100 mg/dose. In another embodiment, thedosage is 10-20 mg/dose. In another embodiment, the dosage is 10-30mg/dose. In another embodiment, the dosage is 10-50 mg/dose. In anotherembodiment, the dosage is 10-80 mg/dose. In another embodiment, thedosage is 10-100 mg/dose.

In another embodiment, the dosage is a daily dose. In anotherembodiment, the dosage is a weekly dose. In another embodiment, thedosage is a monthly dose. In another embodiment, the dosage is an annualdose. In another embodiment, the dose is one is a series of a definednumber of doses. In another embodiment, the dose is a one-time dose. Asdescribed below, in another embodiment, an advantage of RNA,oligoribonucleotide, or polyribonucleotide molecules of the presentinvention is their greater potency, enabling the use of smaller doses.

In another embodiment, the present invention provides a method forproducing a recombinant protein, comprising contacting an in vitrotranslation apparatus with an in vitro-synthesized oligoribonucleotide,the in vitro-synthesized oligoribonucleotide comprising a pseudouridineor a modified nucleoside, thereby producing a recombinant protein.

In another embodiment, the present invention provides a method forproducing a recombinant protein, comprising contacting an in vitrotranslation apparatus with an in vitro-transcribed RNA molecule of thepresent invention, the in vitro-transcribed RNA molecule comprising apseudouridine or a modified nucleoside, thereby producing a recombinantprotein.

In another embodiment, the present invention provides an in vitrotranscription apparatus, comprising: an unmodified nucleotide, anucleotide containing a pseudouridine or a modified nucleoside, and apolymerase. In another embodiment, the present invention provides an invitro transcription kit, comprising: an unmodified nucleotide, anucleotide containing a pseudouridine or a modified nucleoside, and apolymerase. Each possibility represents a separate embodiment of thepresent invention.

In another embodiment, the in vitro translation apparatus comprises areticulocyte lysate. In another embodiment, the reticulocyte lysate is arabbit reticulocyte lysate.

In another embodiment, the present invention provides a method ofreducing an immunogenicity of an oligoribonucleotide molecule or RNAmolecule, the method comprising the step of replacing a nucleotide ofthe oligoribonucleotide molecule or RNA molecule with a modifiednucleotide that contains a modified nucleoside or a pseudouridine,thereby reducing an immunogenicity of an oligoribonucleotide molecule orRNA molecule.

In another embodiment, the present invention provides a method ofreducing an immunogenicity of a gene-therapy vector comprising apolyribonucleotide molecule or RNA molecule, the method comprising thestep of replacing a nucleotide of the polyribonucleotide molecule or RNAmolecule with a modified nucleotide that contains a modified nucleosideor a pseudouridine, thereby reducing an immunogenicity of a gene-therapyvector.

In another embodiment, the present invention provides a method ofenhancing in vitro translation from an oligoribonucleotide molecule orRNA molecule, the method comprising the step of replacing a nucleotideof the oligoribonucleotide molecule or RNA molecule with a modifiednucleotide that contains a modified nucleoside or a pseudouridine,thereby enhancing in vitro translation from an oligoribonucleotidemolecule or RNA molecule.

In another embodiment, the present invention provides a method ofenhancing in vivo translation from a gene-therapy vector comprising apolyribonucleotide molecule or RNA molecule, the method comprising thestep of replacing a nucleotide of the polyribonucleotide molecule or RNAmolecule with a modified nucleotide that contains a modified nucleosideor a pseudouridine, thereby enhancing in vivo translation from agene-therapy vector.

In another embodiment, the present invention provides a method ofincreasing efficiency of delivery of a recombinant protein by a genetherapy vector comprising a polyribonucleotide molecule or RNA molecule,the method comprising the step of replacing a nucleotide of thepolyribonucleotide molecule or RNA molecule with a modified nucleotidethat contains a modified nucleoside or a pseudouridine, therebyincreasing efficiency of delivery of a recombinant protein by a genetherapy vector.

In another embodiment, the present invention provides a method ofincreasing in vivo stability of gene therapy vector comprising apolyribonucleotide molecule or RNA molecule, the method comprising thestep of replacing a nucleotide of the polyribonucleotide molecule or RNAmolecule with a modified nucleotide that contains a modified nucleosideor a pseudouridine, thereby increasing in vivo stability of gene therapyvector.

In another embodiment, the present invention provides a method ofsynthesizing an in vitro-transcribed RNA molecule comprising apseudouridine nucleoside, comprising contacting an isolated polymerasewith a mixture of unmodified nucleotides and the modified nucleotide(Examples 5 and 10).

In another embodiment, in vitro transcription methods of the presentinvention utilize an extract from an animal cell. In another embodiment,the extract is from a reticulocyte or cell with similar efficiency of invitro transcription. In another embodiment, the extract is from anyother type of cell known in the art. Each possibility represents aseparate embodiment of the present invention.

Any of the RNA molecules or oligoribonucleotide molecules of the presentinvention may be used, in another embodiment, in any of the methods ofthe present invention.

In another embodiment, the present invention provides a method ofenhancing an immune response to an antigen, comprising administering theantigen in combination with mitochondrial (mt) RNA (Examples 4 and 8).

In another embodiment, the present invention provides a method ofreducing the ability of an RNA molecule to stimulate a dendritic cell(DC), comprising modifying a nucleoside of the RNA molecule by a methodof the present invention (e.g., see EXAMPLES).

In another embodiment, the DC is a DC1 cell. In another embodiment, theDC is a DC2 cell. In another embodiment, the DC is a subtype of a DC1cell or DC2 cell. Each possibility represents a separate embodiment ofthe present invention.

In another embodiment, the present invention provides a method ofreducing the ability of an RNA molecule to stimulate signaling by TLR3,comprising modifying a nucleoside of the RNA molecule by a method of thepresent invention. In another embodiment, the present invention providesa method of reducing the ability of an RNA molecule to stimulatesignaling by TLR7, comprising modifying a nucleoside of the RNA moleculeby a method of the present invention. In another embodiment, the presentinvention provides a method of reducing the ability of an RNA moleculeto stimulate signaling by TLR8, comprising modifying a nucleoside of theRNA molecule by a method of the present invention. Each possibilityrepresents a separate embodiment of the present invention.

In another embodiment, all of the internucleoside or internucleotidelinkages in the RNA, oligoribonucleotide, or polyribonucleotide moleculeare phosphodiester. In another embodiment, the inter-nucleotide linkagesare predominantly phosphodiester. In another embodiment, most of theinternucleotide linkages are phosphorothioate. In another embodiment,most the inter-nucleotide linkages are phosphodiester. Each possibilityrepresents a separate embodiment of the present invention.

In another embodiment, the percentage of the inter-nucleotide linkagesin the RNA, oligoribonucleotide, or polyribonucleotide molecule that arephosphodiester is above 50%. In another embodiment, the percentage isabove 10%. In another embodiment, the percentage is above 15%. Inanother embodiment, the percentage is above 20%. In another embodiment,the percentage is above 25%. In another embodiment, the percentage isabove 30%. In another embodiment, the percentage is above 35%. Inanother embodiment, the percentage is above 40%. In another embodiment,the percentage is above 45%. In another embodiment, the percentage isabove 55%. In another embodiment, the percentage is above 60%. Inanother embodiment, the percentage is above 65%. In another embodiment,the percentage is above 70%. In another embodiment, the percentage isabove 75%. In another embodiment, the percentage is above 80%. Inanother embodiment, the percentage is above 85%. In another embodiment,the percentage is above 90%. In another embodiment, the percentage isabove 95%.

In another embodiment, a method of the present invention comprisesincreasing the number, percentage, or frequency of modified nucleosidesin the RNA molecule to decrease immunogenicity or increase efficiency oftranslation. As provided herein (e.g., see EXAMPLES), the number ofmodified residues in an RNA, oligoribonucleotide, or polyribonucleotidemolecule determines, in another embodiment, the magnitude of the effectsobserved in the present invention.

In another embodiment, the present invention provides a method forintroducing a recombinant protein into a cell of a subject, comprisingcontacting the subject with an in vitro-transcribed RNA moleculeencoding the recombinant protein, the in vitro-transcribed RNA moleculefurther comprising a pseudouridine or another modified nucleoside,thereby introducing a recombinant protein into a cell of a subject.

In another embodiment, the present invention provides a method fordecreasing TNF-α production in response to a gene therapy vector in asubject, comprising the step of engineering the vector to contain apseudouridine or a modified nucleoside base, thereby decreasing TNF-αproduction in response to a gene therapy vector in a subject.

In another embodiment, the present invention provides a method fordecreasing IL-12 production in response to a gene therapy vector in asubject, comprising the step of engineering the vector to contain apseudouridine or a modified nucleoside base, thereby decreasing IL-12production in response to a gene therapy vector in a subject.

In another embodiment, the present invention provides a method ofreducing an immunogenicity of a gene therapy vector, comprisingintroducing a modified nucleoside into said gene therapy vector, therebyreducing an immunogenicity of a gene therapy vector.

As provided herein, findings of the present invention show that primaryDC have an additional RNA signaling entity that recognizes m5C- andm6A-modified RNA and whose signaling is inhibited by modification of Uresidues.

In another embodiment, an advantage of an RNA, oligoribonucleotide, andpolyribonucleotide molecules of the present invention is that RNA doesnot incorporate to the genome (as opposed to DNA-based vectors). Inanother embodiment, an advantage is that translation of RNA, andtherefore appearance of the encoded product, is instant. In anotherembodiment, an advantage is that the amount of protein generated fromthe mRNA can be regulated by delivering more or less RNA. In anotherembodiment, an advantage is that repeated delivery of purifiedpseudouridine or other modified RNA, oligoribnucleotide, orpolyribonucleotide molecules does not induce an immune response, whereasrepeated delivery of unmodified RNA could induce signaling pathwaysthough RNA sensors.

In another embodiment, an advantage is lack of immunogenicity, enablingrepeated delivery without generation of inflammatory cytokines. Inanother embodiment, stability of RNA is increased by circularization,decreasing degradation by exonucleases.

In another embodiment, the present invention provides a method oftreating a subject with a disease that comprises an immune responseagainst a self-RNA molecule, comprising administering to the subject anantagonist of a TLR-3 molecule, thereby treating a subject with adisease that comprises an immune response against a self-RNA molecule.

In another embodiment, the present invention provides a method oftreating a subject with a disease that comprises an immune responseagainst a self-RNA molecule, comprising administering to the subject anantagonist of a TLR-7 molecule, thereby treating a subject with adisease that comprises an immune response against a self-RNA molecule.

In another embodiment, the present invention provides a method oftreating a subject with a disease that comprises an immune responseagainst a self-RNA molecule, comprising administering to the subject anantagonist of a TLR-8 molecule, thereby treating a subject with adisease that comprises an immune response against a self-RNA molecule.

In another embodiment, the disease that comprises an immune responseagainst a self-RNA molecule is an auto-immune disease. In anotherembodiment, the disease is systemic lupus erythematosus (SLE). Inanother embodiment, the disease is another disease known in the art thatcomprises an immune response against a self-RNA molecule. Eachpossibility represents a separate embodiment of the present invention.

In another embodiment, the present invention provides a kit comprising areagent utilized in performing a method of the present invention. Inanother embodiment, the present invention provides a kit comprising acomposition, tool, or instrument of the present invention.

In another embodiment, the present invention provides a kit formeasuring or studying signaling by a TLR-3, TLR-7 and TLR-8 receptor, asexemplified in Example 7.

In another embodiment, a treatment protocol of the present invention istherapeutic. In another embodiment, the protocol is prophylactic. Eachpossibility represents a separate embodiment of the present invention.

In one embodiment, the phrase “contacting a cell” or “contacting apopulation” refers to a method of exposure, which can be direct orindirect. In one method such contact comprises direct injection of thecell through any means well known in the art, such as microinjection. Inanother embodiment, supply to the cell is indirect, such as viaprovision in a culture medium that surrounds the cell, or administrationto a subject, or via any route known in the art. In another embodiment,the term “contacting” means that the molecule of the present inventionis introduced into a subject receiving treatment, and the molecule isallowed to come in contact with the cell in vivo. Each possibilityrepresents a separate embodiment of the present invention.

Methods for quantification of reticulocyte frequency and for measuringEPO biological activity are well known in the art, and are described,for Example, in Ramos, A S et al (Biological evaluation of recombinanthuman erythropoietin in pharmaceutical products. Braz J Med Biol Res36:1561). Each method represents a separate embodiment of the presentinvention.

Compositions of the present invention can be, in another embodiment,administered to a subject by any method known to a person skilled in theart, such as parenterally, paracancerally, transmucosally,transdermally, intramuscularly, intravenously, intra-dermally,subcutaneously, intraperitoneally, intraventricularly, intra-cranially,intra-vaginally or intra-tumorally.

In another embodiment of methods and compositions of the presentinvention, the compositions are administered orally, and are thusformulated in a form suitable for oral administration, i.e. as a solidor a liquid preparation. Suitable solid oral formulations includetablets, capsules, pills, granules, pellets and the like. Suitableliquid oral formulations include solutions, suspensions, dispersions,emulsions, oils and the like. In another embodiment of the presentinvention, the active ingredient is formulated in a capsule. Inaccordance with this embodiment, the compositions of the presentinvention comprise, in addition to the active compound and the inertcarrier or diluent, a hard gelating capsule.

In other embodiments, the pharmaceutical compositions are administeredby intravenous, intra-arterial, or intra-muscular injection of a liquidpreparation. Suitable liquid formulations include solutions,suspensions, dispersions, emulsions, oils and the like. In anotherembodiment, the pharmaceutical compositions are administeredintravenously and are thus formulated in a form suitable for intravenousadministration. In another embodiment, the pharmaceutical compositionsare administered intra-arterially and are thus formulated in a formsuitable for intra-arterial administration. In another embodiment, thepharmaceutical compositions are administered intra-muscularly and arethus formulated in a form suitable for intra-muscular administration.

In another embodiment, the pharmaceutical compositions are administeredtopically to body surfaces and are thus formulated in a form suitablefor topical administration. Suitable topical formulations include gels,ointments, creams, lotions, drops and the like. For topicaladministration, the compositions or their physiologically toleratedderivatives are prepared and applied as solutions, suspensions, oremulsions in a physiologically acceptable diluent with or without apharmaceutical carrier.

In another embodiment, the composition is administered as a suppository,for example a rectal suppository or a urethral suppository. In anotherembodiment, the pharmaceutical composition is administered bysubcutaneous implantation of a pellet. In another embodiment, the pelletprovides for controlled release of agent over a period of time.

In another embodiment, the active compound is delivered in a vesicle,e.g. a liposome (see Langer, Science 249: 1527-1533 (1990); Treat etal., in Liposomes in the Therapy of Infectious Disease and Cancer,Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989);Lopez-Berestein, ibid., pp. 317-327; see generally ibid).

As used herein “pharmaceutically acceptable carriers or diluents” arewell known to those skilled in the art. The carrier or diluent may bemay be, in various embodiments, a solid carrier or diluent for solidformulations, a liquid carrier or diluent for liquid formulations, ormixtures thereof.

In another embodiment, solid carriers/diluents include, but are notlimited to, a gum, a starch (e.g. com starch, pregeletanized starch), asugar (e.g., lactose, mannitol, sucrose, dextrose), a cellulosicmaterial (e.g. microcrystalline cellulose), an acrylate (e.g.polymethylacrylate), calcium carbonate, magnesium oxide, talc, ormixtures thereof.

In other embodiments, pharmaceutically acceptable carriers for liquidformulations may be aqueous or non-aqueous solutions, suspensions,emulsions or oils. Examples of non-aqueous solvents are propyleneglycol, polyethylene glycol, and injectable organic esters such as ethyloleate. Aqueous carriers include water, alcoholic/aqueous solutions,emulsions or suspensions, including saline and buffered media. Examplesof oils are those of petroleum, animal, vegetable, or synthetic origin,for example, peanut oil, soybean oil, mineral oil, olive oil, sunfloweroil, and fish-liver oil.

Parenteral vehicles (for subcutaneous, intravenous, intra-arterial, orintramuscular injection) include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's and fixedoils. Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers such as those based on Ringer's dextrose, andthe like. Examples are sterile liquids such as water and oils, with orwithout the addition of a surfactant and other pharmaceuticallyacceptable adjuvants. In general, water, saline, aqueous dextrose andrelated sugar solutions, and glycols such as propylene glycols orpolyethylene glycol are preferred liquid carriers, particularly forinjectable solutions. Examples of oils are those of petroleum, animal,vegetable, or synthetic origin, for example, peanut oil, soybean oil,mineral oil, olive oil, sunflower oil, and fish-liver oil.

In another embodiment, the compositions further comprise binders (e.g.acacia, cornstarch, gelatin, carbomer, ethyl cellulose, guar gum,hydroxypropyl cellulose, hydroxypropyl methyl cellulose, povidone),disintegrating agents (e.g. cornstarch, potato starch, alginic acid,silicon dioxide, croscarmelose sodium, crospovidone, guar gum, sodiumstarch glycolate), buffers (e.g., Tris-HCI, acetate, phosphate) ofvarious pH and ionic strength, additives such as albumin or gelatin toprevent absorption to surfaces, detergents (e.g., Tween 20, Tween 80,Pluronic F68, bile acid salts), protease inhibitors, surfactants (e.g.sodium lauryl sulfate), permeation enhancers, solubilizing agents (e.g.,glycerol, polyethylene glycerol), anti-oxidants (e.g., ascorbic acid,sodium metabisulfite, butylated hydroxyanisole), stabilizers (e.g.hydroxypropyl cellulose, hyroxypropylmethyl cellulose), viscosityincreasing agents (e.g. carbomer, colloidal silicon dioxide, ethylcellulose, guar gum), sweeteners (e.g. aspartame, citric acid),preservatives (e.g., Thimerosal, benzyl alcohol, parabens), lubricants(e.g. stearic acid, magnesium stearate, polyethylene glycol, sodiumlauryl sulfate), flow-aids (e.g. colloidal silicon dioxide),plasticizers (e.g. diethyl phthalate, triethyl citrate), emulsifiers(e.g. carbomer, hydroxypropyl cellulose, sodium lauryl sulfate), polymercoatings (e.g., poloxamers or poloxamines), coating and film formingagents (e.g. ethyl cellulose, acrylates, polymethacrylates) and/oradjuvants. Each of the above excipients represents a separate embodimentof the present invention.

In another embodiment, the pharmaceutical compositions provided hereinare controlled-release compositions, i.e. compositions in which thecompound is released over a period of time after 15 administration.Controlled- or sustained-release compositions include formulation inlipophilic depots (e.g. fatty acids, waxes, oils). In anotherembodiment, the composition is an immediate-release composition, i.e. acomposition in which the entire compound is released immediately afteradministration.

In another embodiment, molecules of the present invention are modifiedby the covalent attachment of water-soluble polymers such aspolyethylene glycol, copolymers of polyethylene glycol and polypropyleneglycol, carboxymethyl cellulose, dextran, polyvinyl alcohol,polyvinylpyrrolidone or polyproline. The modified compounds are known toexhibit substantially longer half-lives in blood following intravenousinjection than do the corresponding unmodified compounds (Abuchowski etal., 1981; Newmark et al., 1982; and Katre et al., 1987). Suchmodifications also increase, in another embodiment, the compound'ssolubility in aqueous solution, eliminate aggregation, enhance thephysical and chemical stability of the compound, and greatly reduce theimmunogenicity and reactivity of the compound. As a result, the desiredin vivo biological activity may be achieved by the administration ofsuch polymer-compound abducts less frequently or in lower doses thanwith the unmodified compound.

An active component is, in another embodiment, formulated into thecomposition as neutralized pharmaceutically acceptable salt forms.Pharmaceutically acceptable salts include the acid addition salts (e.g.,formed with the free amino groups of a polypeptide or antibodymolecule), which are formed with inorganic acids such as, for example,hydrochloric or phosphoric acids, or such organic acids as acetic,oxalic, tartaric, mandelic, and the like. Salts formed from the freecarboxyl groups can also be derived from inorganic bases such as, forexample, sodium, potassium, ammonium, calcium, or ferric hydroxides, andsuch organic bases as isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, procaine, and the like.

Each of the above additives, excipients, formulations and methods ofadministration represents a separate embodiment of the presentinvention.

EXAMPLES

The following experimental protocols were employed in the Examplesprovided below, unless indicated otherwise.

Materials and Methods for Examples 1-3

Cell Culture. Newborn human foreskin fibroblast 1079 cells (Cat#CRL-2097, ATCC, Manassas, Va.) and human IMR90 cells (Cat #CCL-186,ATCC) were cultured in Advanced MEM Medium (Invitrogen, Carlsbad,Calif.) supplemented with 10% heat-inactivated fetal bovine serum (FBS,Hyclone Laboratories, Logan, Utah), 2 mM Glutamax (Invitrogen), 0.1 mMβ-mercaptoethanol (Sigma, St. Louis, Mo.), and Penicillin/Streptomycin(Invitrogen). All cells were grown at 37° C. and 5% CO₂. In someexperiments, human iPS cells that were induced using methods describedherein were maintained on irradiated mouse embryonic fibroblasts (MEFs)(R&D Systems, Minneapolis, Minn.) on 10-cm plates pre-coated with 0.1%gelatin (Millipore, Phillipsburg, N.J.) in DMEM/F12 medium supplementedwith 20% KnockOut serum replacer, 0.1 mM L-glutamine (all fromInvitrogen), 0.1 mM β-mercaptoethanol (Sigma) and 100 ng/ml basicfibroblast growth factor (Invitrogen). In some experiments, human iPScells that were induced using methods described herein were maintainedin MEF-conditioned medium that had been collected as previouslydescribed (Xu et al. 2001).

Constructions of Vectors. The cDNAs for the open reading frames (ORFs)of KLF4, LIN28, NANOG, and OCT4 were PCR amplified from cDNA clones(Open Biosystems, Huntsville, Ala.), cloned into a plasmid vectordownstream of a T7 RNA polymerase promoter (Mackie 1988, Studier andMoffatt 1986) (e.g., various pBluescript™, Agilent, La Jolla, Calif. orpGEM™, Promega, Madison, Wis., vectors) and sequenced. The ORF of SOX2was PCR amplified from a cDNA clone (Invitrogen) and the ORF of c-MYCwas isolated by RT-PCR from HeLa cell total RNA. Both SOX2 and c-MYC ORFwere also cloned into a plasmid vector downstream of a T7 RNA polymerasepromoter and sequenced.

Alternative plasmid vectors containing human open reading frames of(KLF4, LIN28, c-MYC, NANOG, OCT4 and SOX2) were cloned intopBluescriptII. These pBluescriptII vectors where constructed by ligatingthe above open reading frames into the EcoRV (cMyc) or EcoRV/SpeI (KLF4,LIN28, NANOG, OCT4, and SOX2) sites between the 5′ and 3′ Xenopus laevisbeta-globin untranslated regions described (Krieg and Melton 1984).

mRNA Production. The T7 RNA polymerase promoter-containing plasmidcontructs (pT7-KLF4, pT7-LIN28, pT7-c-MYC, pT7-OCT4, pT7-SOX2, orpT7-XBg-KLF4, pT7-XBg-LIN28, pT7-XBg-c-MYC, pT7-XBg-OCT4, andpT7-XBg-SOX2) were linearized with BamHI and pT7-NANOG and pT7-XBg-NANOGwere linearized with Xba I. The mSCRIPT™ mRNA production system(EPICENTRE or CellScript, Madison, Wis., USA) was used to produce mRNAwith a 5′ Cap1 structure and a 3′ Poly (A) tail (e.g., withapproximately 150 A residues), except that pseudouridine-5′-triphosphate(TRILINK, San Diego, Calif.) was used in place ofuridine-5′-triphosphate in the T7 RNA polymerase in vitro transcriptionreactions.

mRNA Purification and Analysis. In some experimental embodiments, themRNA was purified by HPLC, column fractions were collected, and the mRNAfractions were analyzed for purity an immunogenicity as described in“Materials and Methods for Examples 35-38” and/or as described and shownfor FIGS. 22-24. In some experimental embodiments, purified RNApreparations comprising or consisting of mRNAs encoding one or morereprogramming factors which exhibited little or no immunogenicity wereused for the experiments for reprogramming human somatic cells to iPScells.

Reprogramming of Human Somatic Cells on MEFs. 1079 fibroblasts wereplated at 1×10⁵ cells/well of a 6-well dish pre-coated with 0.1% gelatin(Millipore) and grown overnight. The 1079 fibroblasts were transfectedwith equal amounts of each reprogramming factor mRNA (KLF4, LIN28,c-MYC, NANOG, OCT4, and SOX2) using TransIT mRNA transfection reagent(MirusBio, Madison, Wis.). A total of three transfections wereperformed, with one transfection being performed every other day, withmedia changes the day after the first and second transfection. The dayafter the third transfection, the cells were trypsinized and 3.3×10⁵cells were plated in 1079 medium onto 0.1% gelatin pre-coated 10-cmplate seeded with 7.5×10⁵ MEFs the day before. The day after plating thetransfected 1079 fibroblasts onto MEFs, the medium was changed to iPScell medium. The iPS cell medium was changed every day. Eight days afterplating the transfected cells onto MEFs, MEF-conditioned medium wasused. MEF conditioned medium was collected as previously described (Xuet al. 2001). Plates were screened every day for the presence ofcolonies with an iPS morphology using an inverted microscope.

Alternative protocols for reprogramming 1079 and IMR90 fibroblasts onMEFs were also used. MEFs were plated at 1.25×10⁵ cells/well of a 0.1%gelatin pre-coated 6 well dish and incubated overnight in completefibroblast media. 1079 or IMR90 fibroblasts were plated at 3×10⁴cells/well of a 6 well dish seeded with MEFs the previous day and grownovernight at 37° C./5% CO₂. The mScript Kit was then used to generateCap1/poly-adenylated mRNA from the following vectors (pT7-Xβg-KLF4,pT7-Xβg-LIN28, pT7-Xβg-c-MYC, pT7-Xβg-NANOG, pT7-Xβg-OCT4, andpT7-Xβg-SOX2) for use in these daily transfections. All sixreprogramming mRNAs were diluted to 100 ng/μl of each mRNA. Equalmolarity of each mRNA was added together using the following conversionfactors (OCT4 is set at 1 and all of the other mRNAs are multiplied bythese conversion factors to obtain equal molarity in each mRNA mix).KLF=1.32, LIN28=0.58, c-MYC=1.26, NANOG=0.85, OCT4=1, and SOX2=0.88. Toobtain equal molarity of each factor 132 μl of KLF4, 58 μl of LIN28, 126μl of c-MYC, 85 μl of NANOG, 100 μl of OCT4 and 88 μl of SOX2 mRNA (eachat 100 ng/μl) would be added together. A 600 μg total dose fortransfections would mean that 100 ng (using molarity conversions above)of each of six reprogramming mRNAs was used. Trans-IT mRNA transfectionreagent was used to transfect these mRNA doses. For all transfections,mRNA pools were added to 250 μl of either DMEM/F12 media withoutadditives or Advanced MEM media without additives. 5 μl of mRNA boostreagent and 5 μl of TransIT transfection reagent was added to each tubeand incubated at room temp for two minutes before adding thetransfection mix to 2.5 mls of either Advanced MEM media with 10%FBS+100 ng/ml of hFGFb or iPS media containing 100 ng/ml of hFGFb.Transfections were repeated everyday for 10-16 days. The media waschanged 4 hours after each transfection. In some experiments, the cellswere trypsinized and replated onto new MEF plates between 5-8 days afterthe initial transfection. 1079 cells were split ⅙ or 1/12 onto new MEFplates while IMR90 cells were split ⅓ or ⅙ onto new MEF plates.

Reprogramming of Human Somatic Cells in MEF-Conditioned Medium. 1079 orIMR90 fibroblasts were plated at 3×10⁵ cells per 10 cm dishes pre-coatedwith 0.1% gelatin (Millipore) and grown overnight. The 1079 or IMR90fibroblasts were transfected with equal amounts of reprogramming factormRNA (KLF4, LIN28, c-MYC, NANOG, OCT4, and SOX2) using TransIT mRNAtransfection reagent (MirusBio, Madison, Wis.). For each transfection,either 6 μg, 18 μg, or 36 μg of each reprogramming mRNA (KLF4, LIN28,c-MYC, NANOG, OCT4, and SOX2) was used per 10-cm dish. A total of threetransfections were performed, with one transfection being performedevery other day with the medium being changed the day after each of thefirst and second transfections. All transfections were performed inMEF-conditioned medium. The day after the third transfection, the cellswere trypsinized and 3×10⁵ cells were plated on new 10-cm dishespre-coated with 0.1% gelatin (Millipore). The cells were grown inMEF-conditioned medium for the duration of the experiment.

Similar daily mRNA transfections were also performed as described in theprevious section with the only difference being that MEFs were not usedas feeder layers, only MEF conditioned media was used.

Immunoflourescence. The 1079 cells or 1079-derived iPS cell plates werewashed with PBS and fixed in 4% paraformaldehyde in PBS for 30 minutesat room temperature. The iPS cells were then washed 3 times for 5minutes each wash with PBS followed by three washes in PBS+0.1% TritonX-100. The iPS cells were then blocked in blocking buffer (PBS+0.1%Triton, 2% FBS, and 1% BSA) for 1 hour at room temperature. The cellswere then incubated for 2 hours at room temperature with the primaryantibody (mouse anti-human OCT4 Cat #sc-5279, Santa Cruz Biotechnology,Santa Cruz, Calif.), (rabbit anti-human NANOG Cat #3580, rabbitanti-human KLF4 Cat #4038, mouse anti-human LIN28 Cat#5930, rabbitanti-human c-MYC Cat#5605, rabbit anti-human SOX2 Cat#3579, and mouseanti-TRA-1-60 all from Cell Signaling Technology, Beverly, Mass.) at a1:500 dilution in blocking buffer. After washing 5 times in PBS+0.1%Triton X-100, the iPS cells were incubated for 2 hours with theanti-rabbit Alexa Fluor 488 antibody (Cat #4412, Cell SignalingTechnology), anti-mouse FITC secondary (Cat #F5262, Sigma), or ananti-mouse Alexa Fluor 555 (Cat#4409, Cell Signaling Technology) at1:1000 dilutions in blocking buffer. Images were taken on a Nikon TS100Finverted microscope (Nikon, Tokyo, Japan) with a 2-megapixel monochromedigital camera (Nikon) using NIS-elements software (Nikon).

Example 1

This Example describes tests to determine if transfections with mRNAencoding KLF4, LIN28, c-MYC, NANOG, OCT4 and SOX2 resulted in expressionand proper subcellular localization of each respective protein productin newborn fetal foreskin 1079 fibroblasts. The mRNAs used in theexperiments were made with pseudouridine-5′-triphosphate substitutingfor uridine-5′-triphosphate (Kariko et al. 2008). The 1079 fibroblastswere transfected with 4 μg of each mRNA per well of a 6-well dish andimmunofluorescence analysis was performed 24 hours post-transfection.Endogenous KLF4, LIN28, NANOG, OCT4 and SOX2 protein levels wereundetectable by immunoflourescence in untransfected 1079 cells (FIG. 1:B, F, N, R, V). Endogenous levels of c-MYC were relatively high inuntransfected 1079 cells (FIG. 1J). Transfections with mRNAs encodingthe transcription factors, KLF4, c-MYC, NANOG, OCT4, and SOX2 allresulted in primarily nuclear localization of each protein 24 hoursafter mRNA transfections (FIG. 1: D, L, P, T, X). The cytoplasmic mRNAbinding protein, LIN28, was localized to the cytoplasm (FIG. 1: H).

Example 2

Having demonstrated efficient mRNA transfection and proper subcellularlocalization of the reprogramming proteins, this Example describesdevelopment of a protocol for iPS cell generation from somaticfibroblasts. Equal amounts (by weight) of KLF4, LIN28, c-MYC, NANOG,OCT4, and SOX2 mRNAs were transfected into 1079 fibroblasts three times(once every other day). The day after the third transfection, the cellswere plated onto irradiated MEF feeder cells and grown in iPS cellmedium. Six days after plating the 1079 fibroblasts onto irradiatedMEFs, two putative iPS cell colonies became apparent on the 10-cm platetransfected with 3 μg of each reprogramming factor mRNA (KLF4, LIN28,c-MYC, NANOG, OCT4, and SOX2). The colonies were allowed to grow until12 days after the last transfection before they were fixed forimmunofluorescence analysis. The inner cell mass-specific marker NANOGis often used to assay whether iPS cell colonies are truly iPS colonies(Gonzalez et al. 2009, Huangfu et al. 2008). NANOG expression arisingfrom the mRNAs that were transfected 12 days earlier would be negligiblebased on previous reports on the duration of mRNA stability andexpression (Kariko et al. 2008). Staining for NANOG showed that both ofthe two iPS cell colonies were NANOG positive (FIG. 2 B, D, and notshown). The surrounding fibroblasts that were not part of the iPS cellcolony were NANOG negative, suggesting that they were not reprogrammedinto iPS cells.

In a subsequent experiment using the same protocol, both 1079fibroblasts and human IMR90 fibroblasts were transfected with the samereprogramming mRNAs. Multiple colonies were detected as early as 4 daysafter plating the transfected cells on irradiated MEFs. When 6 μg ofeach mRNA (KLF4, LIN28, c-MYC, NANOG, OCT4, and SOX2) were used intransfections in 6-well dishes, 3 putative iPS cell colonies were laterdetected in both cell lines after plating on MEFs in 10-cm plates (FIG.3). In addition to analyzing these colonies for expression of NANOG,TRA-1-60, a more stringent marker of fully reprogrammed iPS cells (Chanet al. 2009), was also used for immunofluorescence analysis. iPScolonies generated from 1079 fibroblasts (FIG. 3 A-F) and from IMR90fibroblasts (FIG. 3 G-I) were positive for both NANOG and TRA-1-60,indicating that these colonies are fully reprogrammed type III iPS cellcolonies. This protocol comprising three transfections of mRNAs encodingall six reprogramming factors and then plating onto MEF feeder cellsresulted in a similar reprogramming efficiency (3-6 iPS colonies per1×10⁶ input cells) as was previously reported by protocols comprisingdelivery of the same reprogramming factors by transfection of anexpression plasmid (Aoi et al. 2008).

Example 3

This Example describes attempts to improve the efficiency ofreprogramming differentiated cells using mRNA. In one approach, aprotocol was used that comprised transfecting 1079 or IMR90 fibroblaststhree times (once every other day) with the mRNAs encoding the sixreprogramming factors in MEF-conditioned medium rather than infibroblast medium and then growing the treated 1079 fibroblasts inMEF-conditioned medium rather than plating them on a MEF feeder layerafter the treatments. At the highest transfection dose utilized (36 μgof each reprogramming factor per 10-cm dish), 208 iPS cell colonies weredetected three days after the final transfection (Figure A-F).Interestingly no iPS cell colonies were detected in the dishestransfected with either 6 or 18 μg of each of the reprogramming factorsat the 3-day timepoint, suggesting that a dose above 18 μg wasimportant, under these conditions, for iPS cell colony formation tooccur within 3 days in MEF-conditioned medium. IMR90 cells showed aneven higher number of iPS cell colonies, with around 200 colonies 8 daysafter the last transfection in the plate transfected with three 6-μgdoses of each of the six reprogramming factor mRNAs and >1000 coloniesin IMR90 cells transfected three times with 18-14 or 36-14 doses of eachof the six reprogramming mRNAs (FIG. 4 G-I). Colonies were visible 3days after the final transfection in 1079 cells, whereas colonies onlybecame visible 6-7 days after the final transfection in IMR90 cells.Therefore, the more mature colonies derived from the 1079 cells werelarger and denser and were darker in bright-field images compared to theIMR90 colonies (FIG. 4). All of the colonies on the 1079 platetransfected three times with 36 μg of each reprogramming mRNA werepositive for both NANOG and TRA-1-60 8 days after the final mRNAtransfection (Figures A-I). All of the more immature IMR90 iPS colonieswere also positive for both NANOG and TRA-1-60 (FIG. 5 J-O), but showedless robust staining for both markers due to their less dense cellularnature compared to the more mature 1079 colonies (FIG. 5 A-I). Thepresent protocol comprising delivery of the mRNAs into 1079 or IMR90cells in MEF-conditioned medium had a reprogramming efficiency of 200to >1000 colonies per 3×10⁵ input cells. This protocol for inducing iPScells was faster and almost 2-3 orders of magnitude more efficient thanpublished protocols comprising transfecting fibroblasts with DNAplasmids encoding these same six reprogramming factors in fibroblastmedium (Aoi et al. 2008). Still further, this protocol was over 7-40times more efficient than the published protocol comprising delivery ofreprogramming factors with lentiviruses, based on the published datathat lentiviral delivery of reprogramming factors into 1079 newbornfibroblasts, which resulted in approximately 57 iPS cell colonies per6×10⁵ input cells (Aoi et al. 2008). This protocol is also much fasterthan the published methods.

Example 4 Naturally Occurring RNA Molecules Exhibit DifferentialAbilities to Activate Dendritic Cells Materials and Experimental Methods

Plasmids and Reagents

Plasmids pT7T3D-MART-1 and pUNO-hTLR3 were obtained from the ATCC(Manassas, Va.) and InvivoGen (San Diego, Calif.), respectively. pTEVlucwas obtained from Dr Daniel Gallie (UC Riverside), contains pT7-TEV (theleader sequence of the tobacco etch viral genomic RNA)-luciferase-A50,and is described in Gallie, D R et al, 1995. The tobacco etch viral 5′leader and poly(A) tail are functionally synergistic regulators oftranslation. Gene 165:233) pSVren was generated from p2luc (GrentzmannG, Ingram J A, et al, A dual-luciferase reporter system for studyingrecoding signals. RNA 1998; 4(4): 479-86) by removal of the fireflyluciferase coding sequence with BamHI and NotI digestions, end-filling,and religation.

Human TLR3-specific siRNA, pTLR3-sh was constructed by insertingsynthetic ODN encoding shRNA with 20-nt-long homology to human TLR3 (nt703-722, accession: NM_003265) into plasmid pSilencer 4.1-CMV-neo(Ambion, Austin, Tex.). pCMV-hTLR3 was obtained by first cloninghTLR3-specific PCR product (nt 80-2887; Accession NM_003265) intopCRII-TOPO (Invitrogen, Carlsbad, Calif.), then released with Nhe I-HindIII cutting and subcloning to the corresponding sites of pcDNA3.1(Invitrogen). LPS (E. coli 055:B5) was obtained from Sigma Chemical Co,St. Louis, Mo. CpG ODN2006 and R-848 were obtained from InvivoGen.

Cells and Cell Culture

Human embryonic kidney 293 cells (ATCC) were propagated in DMEMsupplemented with glutamine (Invitrogen) and 10% FCS (Hyclone, Ogden,Utah) (complete medium). In all cases herein, “293 cells” refers tohuman embryonic kidney (HEK) 293 cells. 293-hTLR3 cell line wasgenerated by transforming 293 cells with pUNO-hTLR3. Cell lines293-hTLR7, 293-hTLR8 and 293-hTLR9 (InvivoGen) were grown in completemedium supplemented with blasticidin (10 μg/ml) (Invivogen). Cell lines293-ELAM-Iuc and TLR7-293 (M. Lamphier, Eisai Research Institute,Andover Mass.), and TLR3-293 cells were cultured as described (Kariko etal, 2004, mRNA is an endogenous ligand for Toll-like receptor 3. J BiolChem 279: 12542-12550). Cell lines 293, 293-hTLR7 and 293-hTLR8 werestably transfected with pTLR3-sh and selected with G-418 (400 μg/ml)(Invitrogen). Neo-resistant colonies were screened and only those thatdid not express TLR3, determined as lack of IL-8 secretion loin responseto poly(I):(C), were used in further studies. Leukopheresis samples wereobtained from HIV-uninfected volunteers through an IRB-approvedprotocol.

Murine DC Generation

Murine DCs were generated by collecting bone marrow cells from the tibiaand femurs of 6-8-week-old C57BL/6 mice and lysing the red blood cells.Cells were seeded in 6-well plates at 10⁶ cells/well in 2 ml DMEM+10%FCS and 20 ng/ml muGM-CSF (R & D Systems). On day 3, 2 ml of freshmedium with muGM-CSF was added. On day 6, 2 ml medium/well wascollected, and cells were pelleted and resuspended in fresh medium withmuGM-CSF. On day 7 of the culture, the muDC were harvested, washed.

Natural RNA

Mitochondria were isolated from platelets obtained from the Universityof Pennsylvania Blood Bank using a fractionation lyses procedure(Mitochondria isolation kit; Pierce, Rockford, Ill.). RNA was isolatedfrom the purified mitochondria, cytoplasmic and nuclear fractions of 293cells, un-fractioned 293 cells, rat liver, mouse cell line TUBO, andDH5alpha strain of E. coli by Master Blaster® (BioRad, Hercules,Calif.). Bovine tRNA, wheat tRNA, yeast tRNA, E. coli tRNA, poly(A)+mRNAfrom mouse heart and poly(I):(C) were purchased from Sigma, total RNAfrom human spleen and E. coli RNA were purchased from Ambion.Oligoribonucleotide-5′-rnonophosphates were synthesized chemically(Dharmacon, Lafayette, Colo.).

Aliquots of RNA samples were incubated in the presence of Benzonasenuclease (1 U per 5 μl of RNA at 1 microgram per microliter (μg/μ1) for1 h) (Novagen, Madison, Wis.). Aliquots of RNA-730 were digested withalkaline phosphatase (New England Biolabs). RNA samples were analyzed bydenaturing agarose or polyacrylamide gel electrophoresis for qualityassurance. Assays for LPS in RNA preparations using the LimulusAmebocyte Lysate gel clot assay were negative with a sensitivity of 3picograms per milliliter (pg/ml) (University of Pennsylvania, CoreFacility).

HPLC Analysis

Nucleoside monophosphates were separated and visualized via HPLC. Torelease free nucleoside 3′-monophosphates, 5 μg aliquots of RNA weredigested with 0.1 U RNase T2 (Invitrogen) in 10 μl of 50 mM NaOAc and 2mM EDTA buffer (pH 4.5) overnight, then the samples were injected intoan Agilent 1100 HPLC using a Waters Symmetry C 18 column (Waters,Milford, Mass.). At a flow rate of 1 mL/min, a gradient from 100% bufferA (30 mM KH₂PO₄ and 10 mM tetraethylammonium phosphate [PicA reagent,Waters], pH 6.0) to 30% buffer B (acetonitrile) was run over 60 minutes.Nucleotides were detected using a photodiode array at 254 nm. Identitieswere verified by retention times and spectra.

Dendritic Cell Assays

Dendritic cells in 96-well plates (approximately 1.1×10⁵ cells/well)were treated with R-848, Lipofectin®, or Lipofectin®-RNA for 1 h, thenthe medium was changed. At the end of 8 h (unless otherwise indicated),cells were harvested for either RNA isolation or flow cytometry, whilethe collected culture medium was subjected to cytokine ELISA. The levelsof IL-12 (p′70) (BD Biosciences Pharmingen, San Diego, Calif.), IFN-α,TNF-α, and IL-8 (Biosource International, Camarillo, Calif.) weremeasured in supernatants by sandwich ELISA. Cultures were performed intriplicate or quadruplicate and measured in duplicate.

Northern Blot Analysis

RNA was isolated from MDDCs after an 8 h incubation following treatmentas described above. Where noted, cells were treated with 2.5 μg/mlcycloheximide (Sigma) 30 min prior to the stimulation and throughout theentire length of incubation. RNA samples were processed and analyzed onNorthern blots as described (Kariko et al, 2004, ibid) using human TNF-0and GAPDH probes derived from plasmids (pE4 and pHcGAP, respectively)obtained from ATCC.

Results

To determine the immuno-stimulatory potential of different cellular RNAsubtypes, RNA was isolated from different subcellular compartments—i.e.cytoplasm, nucleus and mitochondria. These RNA fractions, as well astotal RNA, tRNA and polyA-tail-selected mRNA, all from mammaliansources, were complexed to Lipofectin® and added to MDDC. Whilemammalian total, nuclear and cytoplasmic RNA all stimulated MDDC, asevidenced by detectable TNF-α secretion, the TNF-α levels were muchlower than those induced by in vitro-synthesized mRNA (FIG. 6).Moreover, mammalian tRNA did not induce any detectable level of TNF-α,while mitochondrial (mt) RNA induced much more TNF-α than the othermammalian RNA subtypes. Bacterial total RNA was also a potent activatorof MDDC; by contrast, bacterial tRNA induced only a low level of TNF-α.tRNA from other sources (yeast, wheat germ, bovine) werenon-stimulatory. Similar results were observed when RNA from othermammalian sources was tested. When RNA samples were digested withBenzonase, which cleaves ssRNA and dsRNA, RNA signaling was abolished inMDDC, verifying that TNF-α secretion was due to the RNA in thepreparations. The activation potentials of the RNA types testedexhibited an inverse correlation with the extent of nucleosidemodification. Similar results were obtained in the experiments describedin this Example for both types of cytokine-generated DC.

These findings demonstrate that the immunogenicity of RNA is affected bythe extent of nucleoside modification, with a greater degree ofmodification tending to decrease immunogenicity.

Example 5 In Vitro Synthesis of RNA Molecules with Modified NucleosidesMaterials and Experimental Methods

In Vitro-Transcribed RNA

Using in vitro transcription assays (MessageMachine and MegaScript kits;Ambion) the following long RNAs were generated by T7 RNA polymerase(RNAP) as described (Kariko et al, 1998, Phosphate-enhanced transfectionof cationic lipid-complexed mRNA and plasmid DNA. Biochim Biophys Acta1369, 320-334) (Note: the names of templates are indicated inparenthesis; the number in the name of the RNA specifies the length):RNA-1866 (Nde I-linearized pTEVluc) encodes firefly luciferase and a 50nt-long polyA-tail. RNA-1571 (Ssp I-linearized pSVren) encodes Renillaluciferase. RNA-730 (Hind III-linearized pT7T3D-MART-1) encodes thehuman melanoma antigen MART-I. RNA-713 (EcoR I-linearized pTIT3D-MART-1)corresponds to antisense sequence of MART-1, RNA497 (Bgl II-linearizedpCMV-hTLR3) encodes a partial 5′ fragment of hTLR3. Sequences of the RNAmolecules are as follows:

RNA-I866: (SEQ ID No: 1)ggaauucucaacacaacauauacaaaacaaacgaaucucaagcaaucaagcauucuacuucuauugcagcaauuuaaaucauuucuuuuaaagcaaaagcaauuuucugaaaauuuucaccauuuacgaacgauagccauggaagacgccaaaaacauaaagaaaggcccggcgccauucuauccucuagaggauggaaccgcuggagagcaacugcauaaggcuaugaagagauacgcccugguuccuggaacaauugcuuuuacagaugcacauaucgaggugaacaucacguacgcggaauacuucgaaauguccguucgguuggcagaagcuaugaaacgauaugggcugaauacaaaucacagaaucgucguaugcagugaaaacucucuucaauucuuuaugccgguguugggcgcguuauuuaucggaguugcaguugcgcccgcgaacgacauuuauaaugaacgugaauugcucaacaguaugaacauuucgcagccuaccguaguguuuguuuccaaaaagggguugcaaaaaauuuugaacgugcaaaaaaaauuaccaauaauccagaaaauuauuaucauggauucuaaaacggauuaccagggauuucagucgauguacacguucgucacaucucaucuaccucccgguuuuaaugaauacgauuuuguaccagaguccuuugaucgugacaaaacaauugcacugauaaugaauuccucuggaucuacuggguuaccuaaggguguggcccuuccgcauagaacugccugcgucagauucucgcaugccagagauccuauuuuuggcaaucaaaucauuccggauacugcgauuuuaaguguuguuccauuccaucacgguuuuggaauguuuacuacacucggauauuugauauguggauuucgagucgucuuaauguauagauuugaagaagagcuguuuuuacgaucccuucaggauuacaaaauucaaagugcguugcuaguaccaacccuauuuucauucuucgccaaaagcacucugauugacaaauacgauuuaucuaauuuacacgaaauugcuucugggggcgcaccucuuucgaaagaagucggggaagcgguugcaaaacgcuuccaucuuccagggauacgacaaggauaugggcucacugagacuacaucagcuauucugauuacacccgagggggaugauaaaccgggcgcggucgguaaaguuguuccauuuuuugaagcgaagguuguggaucuggauaccgggaaaacgcugggcguuaaucagagaggcgaauuaugugucagaggaccuaugauuauguccgguuauguaaacaauccggaagcgaccaacgccuugauugacaaggauggauggcuacauucuggagacauagcuuacugggacgaagacgaacacuucuucauaguugaccgcuugaagucuuuaauuaaauacaaaggauaucagguggcccccgcugaauuggaaucgauauuguuacaacaccccaacaucuucgacgcgggcguggcaggucuucccgacgaugacgccggugaacuucccgccgccguuguuguuuuggagcacggaaagacgaugacggaaaaagagaucguggauuacguggccagucaaguaacaaccgcgaaaaaguugcgcggaggaguuguguuuguggacgaaguaccgaaaggucuuaccggaaaacucgacgcaagaaaaaucagagagauccucauaaaggccaagaagggcggaaaguccaaauuguaaaauguaacucuagaggauccccaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaca. RNA-1571:(SEQ ID No: 2)ggcuagccaccaugacuucgaaaguuuaugauccagaacaaaggaaacggaugauaacugguccgcaguggugggccagauguaaacaaaugaauguucuugauucauuuauuaauuauuaugauucagaaaaacaugcagaaaaugcuguuauuuuuuuacaugguaacgcggccucuucuuauuuauggcgacauguugugccacauauugagccaguagcgcgguguauuauaccagaccuuauugguaugggcaaaucaggcaaaucugguaaugguucuuauagguuacuugaucauuacaaauaucuuacugcaugguuugaacuucuuaauuuaccaaagaagaucauuuuugucggccaugauuggggugcuuguuuggcauuucauuauagcuaugagcaucaagauaagaucaaagcaauaguucacgcugaaaguguaguagaugugauugaaucaugggaugaauggccugauauugaagaagauauugcguugaucaaaucugaagaaggagaaaaaaugguuuuggagaauaacuucuucguggaaaccauguugccaucaaaaaucaugagaaaguuagaaccagaagaauuugcagcauaucuugaaccauucaaagagaaaggugaaguucgucguccaacauuaucauggccucgugaaaucccguuaguaaaaggugguaaaccugacguuguacaaauuguuaggaauuauaaugcuuaucuacgugcaagugaugauuuaccaaaaauguuuauugaaucggacccaggauucuuuuccaaugcuauuguugaaggugccaagaaguuuccuaauacugaauuugucaaaguaaaaggucuucauuuuucgcaagaagaugcaccugaugaaaugggaaaauauaucaaaucguucguugagcgaguucucaaaaaugaacaaaugucgacgggggccccuaggaauuuuuuagggaagaucuggccuuccuacaagggaaggccagggaauuuucuucagagcagaccagagccaacagccccaccagaagagagcuucaggucugggguagagacaacaacucccccucagaagcaggagccgauagacaaggaacuguauccuuuaacuucccucagaucacucuuuggcaacgaccccucgucacaauaaagauaggggggcaacuaaagggaucggccgcuucgagcagacaugauaagauacauugaugaguuuggacaaaccacaacuagaaugcagugaaaaaaaugcuuuauuugugaaauuugugaugcuauugcuuuauuuguaaccauuauaagcugcaauaaacaaguuaacaacaacaauugcauucauuuuauguuucagguucagggggaggugugggagguuuuuuaaagcaaguaaaaccucuacaaaugugguaaaaucgauaaguuuaaacagauccagguggcacuuuucggggaaaugugcgcggaaccccuauuuguuuauuuuucuaaauacauucaaauauguauccgcucaugagacaauaacccugauaaaugcuucaauaau. RNA-730: (SEQ ID No: 3)gggaauuuggcccucgaggccaagaauucggcacgaggcacgcggccagccagcagacagaggacucucauuaaggaagguguccugugcccugacccuacaagaugccaagagaagaugcucacuucaucuaugguuaccccaagaaggggcacggccacucuuacaccacggcugaagaggccgcugggaucggcauccugacagugauccugggagucuuacugcucaucggcuguugguauuguagaagacgaaauggauacagagccuugauggauaaaagucuucauguuggcacucaaugugccuuaacaagaagaugcccacaagaaggguuugaucaucgggacagcaaagugucucuucaagagaaaaacugugaaccugugguucccaaugcuccaccugcuuaugagaaacucucugcagaacagucaccaccaccuuauucaccuuaagagccagcgagacaccugagacaugcugaaauuauuucucucacacuuuugcuugaauuuaauacagacaucuaauguucuccuuuggaaugguguaggaaaaaugcaagccaucucuaauaauaagucaguguuaaaauuuuaguagguccgcuagcaguacuaaucaugugaggaaaugaugagaaauauuaaauugggaaaacuccaucaauaaauguugcaaugcaugauaaaaaaaaaaaaaaaaaaaacugcggccgca.RNA-713 (SEQ ID No: 4)gggaauaagcuugcggccgcaguuuuuuuuuuuuuuuuuuuuaucaugcauugcaacauuuauugauggaguuuucccaauuuaauauuucucaucauuuccucacaugauuaguacugcuagcggaccuacuaaaauuuuaacacugacuuauuauuagagauggcuugcauuuuuccuacaccauuccaaaggagaacauuagaugucuguauaaauucaagcaaaagugugagagaaauaauuucagcaugucucaggugucucgcuggcucuuaaggugaauaaggugguggugacuguucugcagagaguuucucauaagcagguggagcauugggaaccacagguucacaguuuuucucuugaagagacacuuugcugucccgaugaucaaacccuucuugugggcaucuucuuguuaaggcacauugagugccaacaugaagacuuuuauccaucaaggcucuguauccauuucgucuucuacaauaccaacagccgaugagcaguaagacucccaggaucacugucaggaugccgaucccagcggccucuucagccgugguguaagaguggccgugccccuucuugggguaaccauagaugaagugagcaucuucucuuggcaucuuguagggucagggcacaggacaccuuccuuaaugagaguccucugucugcuggcuggccgcgugccucgugccgaauu. RNA-497:(SEQ ID No: 5)gggagacccaagcuggcuagcagucauccaacagaaucaugagacagacuuugccuuguaucuacuuuugggggggccuuuugcccuuugggaugcugugugcauccuccaccaccaagugcacuguuagccaugaaguugcugacugcagccaccugaaguugacucagguacccgaugaucuacccacaaacauaacaguguugaaccuuacccauaaucaacucagaagauuaccagccgccaacuucacaagguauagccagcuaacuagcuuggauguaggauuuaacaccaucucaaaacuggagccagaauugugccagaaacuucccauguuaaaaguuuugaaccuccagcacaaugagcuaucucaacuuucugauaaaaccuuugccuucugcacgaauuugacugaacuccaucucauguccaacucaauccagaaaauuaaaaauaaucccuuugucaagcagaagaauuuaaucacauua.

To obtain modified RNA, the transcription reaction was assembled withthe replacement of one (or two) of the basic NTPs with the correspondingtriphosphate-derivative(s) of the modified nucleotide 5-methylcytidine,5-methyluridine, 2-thiouridine, N⁶-methyladenosine or pseudouridine(TriLink, San Diego, Calif.). In each transcription reaction, all 4nucleotides or their derivatives were present at 7.5 millimolar (mM)concentration. In selected experiments, as indicated, 6 mM m7GpppG capanalog (New England BioLabs, Beverly, Mass.) was also included to obtaincapped RNA. ORN5 and ORN6 were generated using DNA oligodeoxynucleotidetemplates and T7 RNAP (Silencer® siRNA construction kit, Ambion).

Results

To further test the effect of nucleoside modifications onimmunogenicity, an in vitro system was developed for producing RNAmolecules with pseudouridine or modified nucleosides. In vitrotranscription reactions were performed in which 1 or 2 of the 4nucleotide triphosphates (NTP) were substituted with a correspondingnucleoside-modified NTP. Several sets of RNA with different primarysequences ranging in length between 0.7-1.9 kb, and containing eithernone, 1 or 2 types of modified nucleosides were transcribed. ModifiedRNAs were indistinguishable from their non-modified counterparts intheir mobility in denaturing gel electrophoresis, showing that they wereintact and otherwise unmodified (FIG. 7A). This procedure workedefficiently with any of T7, SP6, and T3 phage polymerases, and thereforeis generalizable to a wide variety of RNA polymerases.

These findings provide a novel in vitro system for production of RNAmolecules with modified nucleosides.

Example 6 In Vitro-Transcribed RNA Stimulates Human TLR3, and NucleosideModifications Reduce the Immunogenicity of RNA Materials andExperimental Methods

Parental-293; 293-hTLR7 and 293-hTLR8 cells, all expressingTLR3-specific siRNA, and 293hTLR9, TLR3-293 were seeded into 96-wellplates (5×10⁴ cells/well) and cultured without antibiotics. On thesubsequent day, the cells were exposed to R-848 or RNA complexed toLipofectin® (Invitrogen) as described (Kariko et al, 1998, ibid). RNAwas removed after one hour (h), and cells were further incubated incomplete medium for 7 h. Supernatants were collected for IL-8measurement.

Results

To determine whether modification of nucleosides influences theRNA-mediated activation of TLRs, human embryonic kidney 293 cells werestably transformed to express human TLR3. The cell lines were treatedwith Lipofectin®-complexed RNA, and TLR activation was monitored asindicated 10 by interleukin (IL)-8 release. Several different RNAmolecules were tested. Unmodified, in vitro transcribed RNA elicted ahigh level of IL-8 secretion. RNA containing m6A or s2U nucleosidemodifications, but contrast, did not induce detectable IL-8 secretion(FIG. 7B). The other nucleoside modifications tested (i.e. m5C, m5U, ψ,and m5C/ψ) had a smaller suppressive effect on TLR3 stimulation (FIG.7B). “ψ” refers to pseudouridine.

Thus, nucleoside modifications such as m⁶A S²U, m⁵C, m⁵U, ψ, reduce theimmunogenicity of RNA as mediated by TLR3 signaling.

Example 7 In Vitro-Transcribed RNA Stimulates Human TLR7 and TLR8, andNucleoside Modifications Reduce the Immunogenicity of RNA

To test the possibility that 293 express endogenous TLR3 that interferewith assessing effects of RNA on specific TLR receptors, expression ofendogenous TLR3 was eliminated from the 293-TLR8 cell line by stablytransfecting the cells with a plasmid expressing TLR3-specific shorthairpin (sh)RNA (also known as siRNA). This cell line was used forfurther study, since it did not respond to poly(I):(C), LPS, andCpG-containing oligodeoxynucleotides (ODNs), indicating the absence ofTLR3, TLR4 and TLR9, but did respond to R-848, the cognate ligand ofhuman TLR8 (FIG. 7B). When the 293-hTLR8 cells expressing TLR3-targetedshRNA (293-hTLR8 shRNA-TLR3 cells) were transfected with in vitrotranscribed RNA, they secreted large amounts of IL-8. By contrast, RNAcontaining most of the nucleoside modifications (m⁵C, m⁵U, ψ, and m⁵C/ψ,S²U) eliminated stimulation (no more IL-8 production than the negativecontrol, i.e. empty vector). m6A modification had a variable effect, insome cases eliminating and in other cases reducing IL-8 release (FIG.7B).

The results of this Example and the previous Example show that (a) RNAwith natural phosphodiester inter-nucleotide linkages (e.g. invitro-transcribed RNA) stimulates human TLR3, TLR7 and TLR8; and (b)nucleoside modifications such as m6A, m5C, m5U, s2U and ψ, alone and incombination, reduce the immunogenicity of RNA as mediated by TLR3, TLR7and TLR8 signaling. In addition, these results provide a novel systemfor studying signaling by specific TLR receptors.

Example 8 Nucleoside Modifications Reduce the Immunogenicity of RNA asMediated by TLR7 and TLR8 Signaling

The next set of experiments tested the ability of RNA isolated fromnatural sources to stimulate TLR3, TLR7 and TLR8. RNA from differentmammalian species were transfected into the TLR3, TLR7 andTLR8-expressing 293 cell lines described in the previous Example. Noneof the mammalian RNA samples induced IL-8 secretion above the level ofthe negative control. By contrast, bacterial total RNA obtained from twodifferent E. coli sources induced robust IL-8 secretion in cellstransfected with TLR3, TLR7 and TLR8, but not TLR9 (FIG. 7C). NeitherLPS nor unmethylated DNA (CpG ODN) (the potential contaminants inbacterial RNA isolates) activated the tested TLR3, TLR7 or TLR8.Mitochondrial RNA isolated from human platelets stimulated human TLR8,but not TLR3 or TLR7.

These results demonstrate that unmodified in vitro-transcribed andbacterial RNA are activators of TLR3, TLR7 and TLR8, and mitochondrialRNA stimulates TLR8. In addition, these results confirm the finding thatnucleoside modification of RNA decreases its ability to stimulate TLR3,TLR7 and TLR8.

Example 9 Nucleoside Modifications Reduce the Capacity of RNA to InduceCytokine Secretion and Activation Marker Expression by Dc

Materials and Experimental Methods

DC Stimulation Assays

After 20 h of incubation with RNA, DCs were stained withCD83-phycoerythrin mAb (Research Diagnostics Inc, Flanders, N.J.),HLA-DR-Cy5PE, and CD80 or CD86-fluorescein isothiocyanate mAb andanalyzed on a FACScalibur® flow cytometer using CellQuest® software (BDBiosciences). Cell culture supernatants were harvested at the end of a20 h incubation and subjected to cytokine ELISA. The levels of IL-12(p′70) (BD Biosciences Pharmingen, San Diego, Calif.), IFN-α, and TNF-α(Biosource International, Camarillo, Calif.) were measured insupernatants by ELISA. Cultures were performed in triplicate orquadruplicate, and each sample was measured in duplicate.

Results

The next experiments tested the ability of RNA containing modified orunmodified nucleosides to stimulate cytokine-generated MDDC. Nucleosidemodifications reproducibly diminished the ability of 5 RNA to induceTNF-α and IL-12 secretion by both GM-CSF/1L-4-generated MDDC and(GMCSF)/IFN-α-generated MDDC, in most cases to levels no greater thanthe negative control (FIGS. 8A and B). Results were similar when othersets of RNA with the same base modifications but different primarysequences and lengths were tested, or when the RNA was further modifiedby adding a 5′ cap structure and/or 3′-end polyA-tail or by removing the5′ triphosphate moiety. RNAs of different length and sequence inducedvarying amounts of TNF-α from DC, typically less than a two-folddifference (FIG. 8C).

Next, the assay was performed on primary DC1 and DC2. Primary monocytoid(DCI, BDCA1⁺) and plasmacytoid (DC2, BDCA4⁺) DC were purified fromperipheral blood. Both cell types produced TNF-α when exposed to R-848,but only DC1 responded to poly(I):(C), at a very low level, indicatingan absence of TLR3 activity in DC2. Transfection of in vitro transcriptsinduced TNF-α secretion in both DC1 and DC2, while m5U, ψ ors2U-modified transcripts were not stimulatory (FIG. 8D). In contrast tothe cytokine-generated DC, m5C and m6A modification of RNA did notdecrease its stimulatory capacity in the primary DC1 and DC2.Transcripts with m6A/ψ double modification were non-stimulatory, while amixture of RNA molecules with single type of modification (m6A+ψ) was apotent cytokine inducer. Thus, uridine modification exerted a dominantsuppressive effect on an RNA molecule in cis in primary DC. Theseresults were consistent among all donors tested.

These findings show that in vitro-transcribed RNA stimulates cytokineproduction by DC. In addition, since DC2 do not express TLR3 or TLR8,and m5C and m6A modification of RNA decreased its stimulatory capacityof TLR7, these findings show that primary DC have an additional RNAsignaling entity that recognizes m5C- and m6A-modified RNA and whosesignaling is inhibited by modification of U residues.

As additional immunogenicity indicators, cell surface expression ofCD80, CD83, CD86 and MEW class II molecules, and secretion of TNF-α weremeasured by FACS analysis of MDDC treated with RNA-1571 and its modifiedversions. Modification of RNA with pseudouridine and modifiednucleosides (m5C, m6A, s2U and m6A/ψ) decreased these markers (FIG. 9),confirming the previous findings.

In summary, RNA's capacity to induce DCs to mature and secrete cytokinesdepends on the subtype of DC as well as on the characteristics ofnucleoside modification present in the RNA. An increasing amount ofmodification decreases the immunogenicity of RNA.

Example 10 Suppression of RNA-Mediated Immune Stimulation isProportional to the Number of Modified Nucleosides Present in RNA

Materials and Experimental Methods

Human DC

For cytokine-generated DC, monocytes were purified from PBMC bydiscontinuous Percoll gradient centrifugation. The low density fraction(monocyte enriched) was depleted of B, T, and, NK cells using magneticbeads (Dynal, Lake Success, N.Y.) specific for CD2, CD16, CD19, andCD56, yielding highly purified monocytes as determined by flow cytometryusing anti-CD14 (>95%) or antiCD11c (>98%) mAb.

To generate immature DC, purified monocytes were cultured in AIM Vserum-free medium (Life Technologies), supplemented with GM-CSF (50ng/ml)+IL-4 (100 ng/ml) (R & D Systems, Minneapolis, Minn.) in AIM Vmedium (Invitrogen) for the generation of monocyte-derived DC (MDDC) asdescribed (Weissman, D et al, 2000. J Immunol 165: 4710-4717). DC werealso generated by treatment with GM-CSF (50 ng/ml)+IFN-α (1,000 V/ml) (R& D Systems) to obtain IFN-α MDDC (Santini et al., 2000. Type Iinterferon as a powerful adjuvant for monocyte-derived dendritic celldevelopment and activity in vitro and in Hu-PBL-SCID mice. J Exp Med191: 1777-178).

Primary myeloid and plasmacytoid DCs (DC1 and DC2) were obtained fromperipheral blood using BDCA-1 and BDCA-4 cell isolation kits (MiltenyiBiotec Auburn, Calif.), respectively.

Results

Most of the nucleoside-modified RNA utilized thus far contained one typeof modification occurring in approximately 25% of the total nucleotidesin the RNA (e.g. all the uridine bases). To define the minimal frequencyof particular modified nucleosides that is sufficient to reduceimmunogenicity under the conditions utilized herein, RNA molecules withlimited numbers of modified nucleosides were generated. In the first setof experiments, RNA was transcribed in vitro in the presence of varyingratios of m6A, ψ (pseudouridine) or m5C to their correspondingunmodified NTPs. The amount of incorporation of modified nucleosidephosphates into RNA was expected to be proportional to the ratiocontained in the transcription reaction, since RNA yields obtained withT7 RNAP showed the enzyme utilizes NTPs of m6A, ψ or m5C almost asefficiently as the basic NTPs. To confirm this expectation, RNAtranscribed in the presence of UTP:ψ in a 50:50 ratio was digested andfound to contain UMP and ψ in a nearly 50:50 ratio (FIG. 10A).

RNA molecules with increasing modified nucleoside content weretransfected into MDDC, and TNF-α secretion was assessed. Eachmodification (m6A, ψ and m5C) inhibited TNF-α secretion proportionallyto the fraction of modified bases. Even the smallest amounts of modifiedbases tested (0.2-0.4%, corresponding to 3-6 modified nucleosides per1571 nt molecule), was sufficient to measurably inhibit cytokinesecretion (FIG. 10B). RNA with of 1.7-3.2% modified nucleoside levels(14-29 modifications per molecule) exhibited a 50% reduction ininduction of TNF-α expression. In TLR-expressing 293 cells, a higherpercentage (2.5%) of modified nucleoside content was required to inhibitRNA-mediated signaling events.

Thus, pseudouridine and modified nucleosides reduce the immunogenicityof RNA molecules, even when present as a small fraction of the residues.

In additional experiments, 21-mer oligoribonucleotides (ORN) withphosphodiester inter-nucleotide linkages were synthesized whereinmodified nucleosides (m5C, ψ or 2′-O-methyl-U [Um]) were substituted ina particular position (FIG. 11A). While the unmodified ORN induced TNF-αsecretion, this effect was abolished by the presence of a singlenucleoside modification (FIG. 11B). Similar results were obtained withTLR-7 and TLR-8-transformed 293 cells expressing TLR3-targeted siRNA.

The above results were confirmed by measuring TNF-α mRNA levels in MDDCby Northern blot assay, using both the above 21-mer ORN (ORN1) and31-mer in vitro-synthesized transcripts (ORN5 and ORN6). To amplify thesignal, cycloheximide, which blocks degradation of selected mRNAs, wasadded to some samples, as indicated in the Figure. The unmodified ODNincreased TNF-α mRNA levels, while ORNs containing a single modifiednucleoside were significantly less stimulatory; ORN2-Um exhibited thegreatest decrease TNF-α production (FIG. 11C). Similar results wereobserved in mouse macrophage-like RAW cells and in human DC.

In summary, each of the modifications tested (m6A, m5C, m5U, s2U, ψ and2′-O-methyl) suppressed RNA mediated immune stimulation, even whenpresent as a small fraction of the residues. Further suppression wasobserved when the proportion of modified nucleosides was increased.

Example 11 Pseudouridine-Modification of RNA Reduces its ImmunogenicityIn Vivo

To determine the effect of pseudouridine modification on immunogenicityof RNA in vivo, 0.25 μg RNA) was complexed to Lipofectin® and injectedintra-tracheally into mice, mice were bled 24 h later, and circulatinglevels of TNF-α and IFN-α were assayed from serum samples. Capped,pseudouridine-modified mRNA induced significantly less TNF-α. and IFN-αmRNA than was elicited by unmodified mRNA (FIG. 12A-B).

These results provide further evidence that pseudouridine-modified mRNAis significantly less immunogenic in vivo than unmodified RNA.

Example 12 Pseudouridine-Containing RNA Exhibits Decreased Ability toActivate PRK

Materials and Experimental Methods

PKR Phosphorylation Assays

Aliquots of active PKR agarose (Upstate) were incubated in the presenceof magnesium/ATP coctail (Upstate), kinase buffer and [gamma³²p] ATP mixand RNA molecules for 30 min at 30° C. Unmodified RNA and RNA withnucleoside modification (m5C, pseudouridine, m6A, m5U) and dsRNA weretested. Human recombinant eIF2a (BioSource) was added, and samples werefurther incubated for 5 min, 30° C. Reactions were stopped by addingNuPage LDS sample buffer with reducing reagent (Invitrogen), denaturedfor 10 min, 70° C., and analyzed on 10% PAGE. Gels were dried andexposed to film. Heparin (1 U/μl), a PKR activator, was used as positivecontrol.

Results

To determine whether pseudouridine-containing mRNA activatesdsRNA-dependent protein kinase (PKR), in vitro phosphorylation assayswere performed using recombinant human PKR and its substrate, eIF2α(eukaryotic initiation factor 2 alpha) in the presence of capped,renilla-encoding mRNA (0.5 and 0.05 ng/μ1). mRNA containingpseudouridine (ψ) did not activate PKR, as detected by lack of bothself-phosphorylation of PKR and phosphorylation of eIF2α, while RNAwithout nucleoside modification and mRNA with m⁵C modification activatedPKR (FIG. 13). Thus, pseudouridine modification decreases RNAimmunogenicity.

Example 13 Enhanced Translation of Proteins from Pseudouridine andm⁵C-Containing RNA In Vitro

Materials and Experimental Methods

In Vitro Translation of mRNA in Rabbit Reticulocyte Lysate

In vitro-translation was performed in rabbit reticulocyte lysate(Promega, Madison Wis.). A 9-μl aliquot of the lysate was supplementedwith 1 μl (1 μg) mRNA and incubated for 60 min at 30° C. One μl aliquotwas removed for analysis using firefly and renilla assay systems(Promega, Madison Wis.), and a LUMAT LB 950 luminometer (Berthold/EG&GWallac, Gaithersburg, Md.) with a 10 sec measuring time.

Results

To determine the effect of pseudouridine modification on RNA translationefficiency in vitro, (0.1 μg/μl) uncapped mRNA modified withpseudouridine encoding firefly luciferase was incubated in rabbitreticulocyte lysate for 1 h at 30° C., and luciferase activity wasdetermined. mRNA containing pseudouridine was translated more than2-fold more efficiently than RNA without pseudouridine in rabbitreticulocyte lysates, but not in wheat extract or E. coli lysate (FIG.14), showing that pseudouridine modification increases RNA translationefficiency. Similar results were obtained with m⁵C-modified RNA. When apolyA tail was added to pseudouridine-containing mRNA, a further 10-foldincrease in translation efficiency was observed. (Example 10).

Thus, pseudouridine and m⁵C modification increases RNA translationefficiency, and addition of a polyA tail to pseudouridine-containingmRNA further increases translation efficiency.

Example 14 Enhanced Translation of Proteins from PseudouridineContaining RNA in Cultured Cells

Materials and Experimental Methods

Translation Assays in Cells

Plates with 96 wells were seeded with 5×10⁴ cells per well 1 day beforetransfection. Lipofectin®-mRNA complexes were assembled and addeddirectly to the cell monolayers after removing the culture medium (0.2μs mRNA-0.8 μg lipofectin in 50 μl per well). Cells were incubated withthe transfection mixture for 1 h at 37° C., 5% CO₂ incubator, then themixture was replaced with fresh, pre-warmed medium containing 10% FCS,then cells were analyzed as described in the previous Example.

Results

To determine the effect of pseudouridine modification on RNA translationin cultured cells, 293 cells were transfected with in vitro-transcribed,nucleoside-modified, capped mRNA encoding the reporter protein renilla.Cells were lysed 3 h after initiation of transfection, and levels ofrenilla were measured by enzymatic assays. In 293 cells, pseudouridine-and m5C-modified DNA were translated almost 10 times and 4 times moreefficiently, respectively, than unmodified mRNA (FIG. 15A).

Next, the experiment was performed with primary, bone marrow-derivedmouse DC, in this case lysing the cells 3 h and 8 h after transfection.RNA containing the pseudouridine modification was translated 15-30 timesmore efficiently than unmodified RNA (FIG. 15B).

Similar expression results were obtained using human DC and otherprimary cells and established cell lines, including CHO and mousemacrophage-like RAW cells. In all cell types, pseudouridine modificationproduced the greatest enhancement of the modifications tested.

Thus, pseudouridine modification increased RNA translation efficiency inall cell types tested, including different types of both professionalantigen-presenting cells and non-professional antigen-presenting cells,providing further evidence that pseudouridine modification increases theefficiency of RNA translation.

Example 15 5′ and 3′ Elements Further Enhance the Translation of ψmRNAin Mammalian Cells

To test the effect of additional RNA structural elements on enhancementof translation by pseudouridine modification, a set of fireflyluciferase-encoding ψmRNAs were synthesized that contained combinationsof the following modifications: 1) a unique 5′ untranslated sequence(TEV, a cap independent translational enhancer), 2) cap and 3)polyA-tail. The ability of these modifications to enhance translation ofψmRNA or conventional mRNA was assessed (FIG. 16A). These structuralelements additively enhanced translational efficiency of bothconventional and ψmRNA, with ψmRNA exhibiting greater protein productionfrom all constructs.

Ability of protein expression from the most efficient firefly luciferaseψmRNA construct, capTEVlucA50 (containing TEV, cap, and an extendedpoly(A) tail) was next examined over 24 hours in 293 cells (FIG. 16B).ψmRNA produced more protein at every time point tested and conferredmore persistent luciferase expression than equivalent conventional mRNAconstructs, showing that ψ-modifications stabilize mRNA.

To test whether ψ-modification of mRNA improved translation efficiencyin mammalian cells in situ, caplacZ-ψmRNA constructs with or withoutextended polyA-tails (An) and encoding β-galactosidase (lacZ) weregenerated and used to transfect 293 cells. 24 h after mRNA delivery,significant increases in β-galactosidase levels were detected by X-galvisualization, in both caplacZ and caplacZ-A_(n), compared to thecorresponding control (conventional) transcripts (FIG. 16C). This trendwas observed when either the number of cells expressing detectablelevels of β-galactosidase or the signal magnitude in individual cellswas analyzed.

Example 16 Enhanced Translation of Proteins from PseudouridineContaining RNA In Vivo Materials and Experimental Methods

Intracerebral RNA Injections

All animal procedures were in accordance with the NIH Guide for Care andUse of Laboratory Animals and approved by the Institutional Animal Careand Use Committee. Male Wistar rats (Charles River Laboratories,Wilmington, Mass.) were anesthetized by intraperitoneal injection ofsodium pentobarbital (60 mg/kg body weight). Heads were placed in astereotaxic frame, and eight evenly spaced 1.5 mm diameter burr holeswere made bilaterally (coordinates relative to bregma:anterior/posterior +3, 0, −3, −6 mm; lateral±2.5 mm) leaving the duraintact. Intra-cerebral injections were made using a 25 μl syringe(Hamilton, Reno, Nev.) with a 30 gauge, 1 inch sterile needle (Beckton25 Dickinson Labware, Franklin Lakes, N.J.) which was fixed to a largeprobe holder and stereotactic arm. To avoid air space in the syringe,the needle hub was filled with 55 μl complex before the needle wasattached, and the remainder of the sample was drawn through the needle.Injection depth (2 mm) was determined relative to the surface of thedura, and 4 μl complex (32 ng mRNA) was administered in a single, rapidbolus infusion. 3 hours (h) later, rats were euthanized with halothane,and brains were removed into chilled phosphate buffered saline.

Injection of RNA into Mouse Tail Vein

Tail veins of female BALB/c mice (Charles River Laboratories) wereinjected (bolus) with 60 μl Lipofectin®-complexed RNA (0.26 μg). Organswere removed and homogenized in luciferase or Renilla lysis buffer inmicrocentrifuge tubes using a pestle. Homogenates were centrifuged, andsupernatants were analyzed for activity.

Delivery of RNA to the Lung

Female BALB/c mice were anaesthetized using ketamine (100 mg/kg) andxylasine (20 mg/kg). Small incisions were made in the skin adjacent tothe trachea. When the trachea was exposed, 501-11 ofLipofectin®-complexed RNA (0.2 μg) was instilled into the tracheatowards the lung. Incisions were closed, and animals allowed to recover.3 hours after RNA delivery, mice were sacrificed by cervical dislocationand lungs were removed, homogenized in luciferase or Renilla lysisbuffer (250 μl), and assayed for activity. In a different set ofanimals, blood samples (100 μl/animal) were collected from tail veins,clotted, and centrifuged. Serum fractions were used to determine levelsof TNF and IFNα by ELISA as described in the Examples above, usingmouse-specific antibodies.

Results

To determine the effect of pseudouridine modification on RNA translationin vivo, each hemisphere of rat brain cortexes was injected with eithercapped, renilla-encoding pseudouridine modified RNA or unmodified RNA,and RNA translation was measured. Pseudouridine-modified RNA wastranslated significantly more efficiently than unmodified RNA (FIG.17A).

Next, expression studies were performed in mice. Fireflyluciferase-encoding mRNAs because no endogenous mammalian enzymeinterferes with its detection. Transcripts (unmodified and ψmRNA) wereconstructed with cap, TEV (capTEVA₅₀) and extended (−200 nt) poly(A)tails. 0.25 μg RNA Lipofectin®-complexed was injected into mice(intravenous (i.v.) tail vein). A range of organs were surveyed forluciferase activity to determine the optimum measurement site.Administration of 0.3 μg capTEVlucAn ψmRNA induced high luciferaseexpression in spleen and moderate expression in bone marrow, but littleexpression in lung, liver, heart, kidney or brain (FIG. 17B). Insubsequent studies, spleens were studied.

Translation efficiencies of conventional and ψmRNA (0.015 mg/kg; 0.3μg/animal given intravenously) were next compared in time courseexperiments. Luciferase activity was readily detectable at 1 h, peakedat 4 h and declined by 24 h following administration of eitherconventional or ψmRNA, but at all times was substantially greater inanimals given ψmRNA (FIG. 17C, left panel). By 24 h, only animalsinjected with ψmRNA demonstrated detectable splenic luciferase activity(4-fold above background). A similar relative pattern of expression(between modified and unmodified mRNA) was obtained when mRNAs encodingRenilla luciferase (capRen with or without ψ modifications) wereinjected into the animals instead of firefly luciferase, or whenisolated mouse splenocytes were exposed to mRNA in culture.

In the next experiment, 0.25 μg mRNA-Lipofectin® was delivered to mouselungs by intratracheal injection. Capped, pseudouridine-modified RNA wastranslated more efficiently than capped RNA without pseudouridinemodification (FIG. 17D).

Thus, pseudouridine modification increases RNA translation efficiency invitro, in cultured cells, and in vivo-in multiple animal models and bymultiple routes of administration, showing its widespread application asa means of increasing the efficiency of RNA translation.

Example 17 Pseudouridine Modification Enhances RNA Stability In Vivo

Northern analyses of splenic RNA at 1 and 4 h post injection in theanimals from the previous Example revealed that the administered mRNAs,in their intact and partially degraded forms, were readily detectable(FIG. 17C, right panel). By contrast, at 24 h, unmodified capTEVlucAnmRNA was below the level of detection, while capTEVlucAn ψmRNA, thoughpartially degraded, was still clearly detectable. Thus, ψmRNA is morestably preserved in vivo than control mRNA.

To test whether in vivo protein production is quantitatively dependenton the concentration of intravenously-delivered mRNA, mRNAs wereadministered to mice at 0.015-0.150 mg/kg (0.3-3.0 μg capTEVlucAn peranimal) and spleens were analyzed 6 hours later as described above.Luciferase expression correlated quantitatively with the amount ofinjected RNA (FIG. 18) and at each concentration.

These findings confirm the results of Example 15, demonstrating thatψmRNA is more stable than unmodified RNA. Further immunogenicity ofψ-mRNA was less than unmodified RNA, as described herein above (FIG. 12and FIG. 17C, right panel).

To summarize Examples 16-17, the 3 advantages of ψ-mRNA compared withconventional mRNA (enhanced translation, increased stability and reducedimmunogenicity) observed in vitro are also observed in vivo.

Example 18 ψmRNA Delivered Via the Respiratory Tract Behaves Similarlyto Intravenously Administered mRNA

To test the ability of ψmRNA to be delivered by inhalation, Lipofectin®-or PEI-complexed mRNAs encoding firefly luciferase were delivered tomice by the intratracheal route, wherein a needle was placed into thetrachea and mRNA solution sprayed into the lungs. Similar to intravenousdelivery, significantly greater luciferase expression was observed withψmRNA compared to unmodified mRNA (FIG. 19), although significantly lessprotein was produced with the intratracheal as compared to theintravenous routes. Unmodified mRNA administered by the intratrachealroute was associated with significantly higher concentrations ofinflammatory cytokines (IFN-α and TNF-α) compared with vehicle controls,while ψmRNA was not (FIG. 19).

Thus, ψmRNA can be delivered by inhalation without activating the innateimmune response.

Example 19 Delivery of EPO-ψmRNA to 293 Cells

ψmRNA was generated from a plasmid containing the human EPO cDNA. When0.25 μg of EPO-ψmRNA was transfected into 10⁶ cultured 293 cells,greater than 600 mU/ml of EPO protein was produced. Thus, modified RNAmolecules of the present invention are efficacious at deliveringrecombinant proteins to cells.

Example 20 Preparation of Improved EPO-Encoding ψmRNA Constructs

Materials and Experimental Methods

The EPO coding sequence is cloned using restriction enzyme techniques togenerate 2 new plasmids, pTEV-EPO and pT7TS-EPO, that are used astemplates for EPO-ψmRNA production. EPO-ψmRNAs are produced from thesetemplates by in vitro transcription (MessageMachine® and MegaScript®kits; Ambion) using T7 RNA polymerase (RNAP), incorporating nucleosidesat equimolar (7.5 mM) concentrations. To incorporate thenucleoside-modifications, ψ triphosphate (TriLink, San Diego, Calif.)replaces UTP in the transcription reaction. To ensure capping of theψmRNA, a non-reversible cap-analog, 6 mM 3′-O-Me-m7GpppG (New EnglandBioLabs, Beverly, Mass.) is also included. The ψmRNAs are poly(A)-tailedin a reaction of ˜1.5 μg/μl RNA, 5 mM ATP, and 60 U/μ1 yeast poly(A)polymerase (USB, Cleveland, Ohio) mixed at 30° C. for 3 to 24 h. Qualityof ψmRNAs is assessed by denaturing agarose gel electrophoresis. Assaysfor LPS in mRNA preparations using the Limulus Amebocyte Lysate gel clotassay with a sensitivity of 3 pg/ml are also performed.

Results

The proximal 3′-untranslated region (3′UTR) of EPO-ψmRNA preserves a ˜90nt-long pyrimidine-rich stabilizing element from the nascent EPO mRNA,which stabilizes EPO mRNA by specific association with a ubiquitousprotein, erythropoietin mRNA-binding protein (ERBP). To maximize thestability of EPO-ψmRNA, 2 alterations are incorporated into the EPOplasmid to improve the stability and translational efficiency of thetranscribed mRNA: 1) A 5′UTR sequence of the tobacco etch virus (TEV) isincorporated upstream of the EPO coding sequence to generate pTEV-EPO.2) A plasmid, pT7TS-EPO, is generated, wherein the EPO cDNA is flankedby sequences corresponding to 5′ and 3′ UTRs of Xenopus beta-globinmRNA.

In addition, the length of the poly(A) tail during the production ofψmRNA from these plasmid templates is extended, by increasing theincubation period of the poly(A) polymerase reaction. The longer poly(A)tail diminishes the rate at which ψmRNA degrades during translation.

These improvements result in enhanced translation efficiency in vivo,thus minimizing the therapeutic dose of the final product.

Example 21 In Vitro Analysis of Protein Production from EPO mRNAConstructs

Materials and Experimental Methods

Preparation of Mammalian Cells.

Human embryonic kidney 293 cells (ATCC) are propagated in DMEMsupplemented with glutamine (Invitrogen) and 10% FCS (Hyclone, Ogden,Utah) (complete medium). Leukopheresis samples are obtained fromHIV-uninfected volunteers through an IRB-approved protocol. DCs areproduced as described above and cultured with GM-CSF (50 ng/ml)+IL-4(100 ng/ml) (R & D Systems) in AIM V Medium® (Invitrogen).

Murine spleen cells and DC are obtained by published procedures.Briefly, spleens from BALB/c mice are aseptically removed and mincedwith forceps in complete medium. Tissue fragments are sedimented bygravity and the single cell suspension washed and lysed with AKC lysisbuffer (Sigma). Murine DCs are derived from bone marrow cells collectedfrom femurs and tibia of 6-9-weekold BALB/c mice. Cells are cultured inDMEM containing 10% FCS (Invitrogen) and 50 ng/ml muGM-CSF (R&D) andused on day 7.

Transfection of Cells and Detection of EPO and Pro-InflammatoryCytokines

Transfections are performed with Lipofectin in the presence of phosphatebuffer, an effective delivery method for splenic and in vitro cellexpression. EPO-ψmRNA (0.25 μg/well; 100,000 cells) is added to eachcell type in triplicate for 1 hour, and supernatant replaced with freshmedium. 24 hours later, supernatant is collected for ELISA measurementof EPO, IFN-α or β and TNF-α.

Results

To evaluate the impact of unique UTRs on enhancement of ψmRNAtranslational efficiency, EPO-ψmRNA containing, or not containing, eachimprovement (5′ TEV element, (β-globin 5′ and 3′ UTRs) with long poly(A)tails are tested for in vitro protein production and in vitro immuneactivation using EPO mRNA containing conventional nucleosides ascontrols. Efficiency of protein production from each mRNA is assessed inmammalian cell lines, (HEK293, CHO), human and murine primary DCs, andspleen cells for each mRNA. Measurement of total EPO produced in allcell types and immunogenicity (supernatant-associated proinflammatorycytokines) in primary cells is evaluated. The mRNA construct thatdemonstrates the optimum combination of high EPO production (in 1 ormore cell types) and low cytokine elicitation is used in subsequentstudies. Improvements in 5′ and 3′UTRs of EPO-ψmRNA and longer poly(A)tails result in an estimated 2-10-fold enhancement in translationefficiency, with no increase in immunogenicity.

Example 22 Characterization of EPO Production and Biological Response toEPO-ψmRNA In Vivo

Materials and Experimental Methods

Administration of EPO-ψmRNA to Mice.

All animal studies described herein are performed in accordance with theNIH Guide for Care and Use of Laboratory Animals and approved by theInstitutional Animal Care and Use Committee of the University ofPennsylvania. Female BALB/c mice (n=5 per experimental condition; 6weeks, 18-23 g; Charles River Laboratories) are anesthetized using 3.5%halothane in a mixture of N₂O and O₂ (70:30), then halothane reduced to1% and anesthesia maintained using a nose mask. Animal body temperaturesare maintained throughout the procedure using a 37° C. warmed heatingpad. EPO-ψmRNA-lipofectin complexes (constructed by mixing varyingamounts of nucleic acid with 1 μl lipofectin in 60 μl final volume areinjected into the lateral tail vein. Blood samples are collected 3 timesa day for 3 days post mRNA injection during the time-course study, at 1optimal time point in dose-response studies, and daily from days 2-6 instudies for reticulocytosis.

Determination of Reticulocytes by Flow Cytometry.

Whole blood samples are stained using Retic-COUNT reagent (BDDiagnostics) and data events acquired on a FACScan flow cytometer. Redblood cells (RBCs) are selected by forward and side scatter propertiesand analyzed for uptake of Thiazole Orange. Cells stained withRetic-COUNT reagent are detected by fluorescence and reticulocytesexpressed as the percentage of total RBC. At least 50,000 events arecounted per sample.

Results

To optimize production of biologically functional human EPO protein(hEPO) in response to EPO-encoding mRNA, the following studies areperformed:

Time course of EPO production after a single injection of EPO-ψmRNA.Following intravenous administration of 1 μg EPO-ψmRNA, hEPO is measuredserially from 1-96 h after EPO-ψmRNA administration by ELISA, todetermine the half-life of EPO protein in the serum. This half-life is aresult of both the half-life of EPO protein and the functional half-lifeof the EPO-ψmRNA. The resulting optimal time point for measuring EPOprotein after EPO-ψmRNA administration is utilized in subsequentstudies.

Dose-response of EPO production after a single injection of EPO-ψmRNA.To determine the correlation between the amount of EPO protein producedand the amount of EPO-ψmRNA administered, increasing concentrations ofEPO-ψmRNA (0.01 to 1 μg/animal) are administered and EPO is measured atthe optimal time point.

Relationship between hEPO production and reticulocytosis. To measure theeffect of EPO-ψmRNA on a biological correlate of EPO activity, flowcytometry is used to determine reticulocyte frequency in blood). Flowcytometry has a coefficient of variation of <3%. Mice receive a singledose of EPO-ψmRNA, and blood is collected from mice daily from days 2-6.The relationship between EPO-ψmRNA dose and reticulocyte frequency isthen evaluated at the time point of maximal reticulocytosis. The dose ofEPO-ψmRNA that leads to at least a 5% increase in reticulocyte count isused in subsequent studies. Serum hEPO concentrations in mice of anestimated 50 mU/ml and/or an increase in reticulocyte frequency of anestimated 5% are obtained.

Example 23 Measuring Immune Responses to EPO-ψmRNA In Vivo

Materials and Experimental Methods

Detection of Cytokines in Plasma.

Serum samples obtained from blood collected at different times duringand after 7 daily lipofectin-complexed mRNA administrations are analyzedfor mouse IFN-α, TNF-α, and IL-12 using ELISA kits.

Northern Blot Analysis.

Aliquots (2.0 μg) of RNA samples isolated from spleen are separated bydenaturing 1.4% agarose gel electrophoresis, transferred to chargedmembranes (Schleicher and Schuell) and hybridized in MiracleHyb®(Stratagene). Membranes are probed for TNF-α, down-stream IFN signalingmolecules (e.g. IRF7, IL-12 p35 and p40, and GAPDH) and other markers ofimmune activation. Specificity of all probes is confirmed by sequencing.To probe the membranes, 50 ng of DNA is labeled using Redivue[alpha-³²P] dCTP® (Amersham) with a random prime labeling kit (Roche).Hybridized membranes are exposed to Kodak BioMax MS film using an MSintensifier screen at −70° C.

Histopathology.

Spleens from EPO-ψmRNA-treated and positive and negative control-treatedmice are harvested, fixed, sectioned, stained with hematoxylin and eosinand examined by a veterinary pathologist for signs of immune activation.

Results

To confirm the reduced immunogenicity of RNA molecules of the presentinvention, mice (n=5) receive daily doses of EPO-ψmRNA for 7 days, thenare evaluated for immune-mediated adverse events, as indicated by serumcytokine concentrations, splenic expression of mRNAs encodinginflammatory proteins, and pathologic examination. Maximum administereddoses are 3 μg or 5× the effective single dose as determined above.Unmodified mRNA and Lipofectin® alone are used as positive and negativecontrols, respectively.

These studies confirm the reduced immunogenicity of RNA molecules of thepresent invention.

Example 24 FURTHER IMPROVEMENT OF EPO-ψmRNA DELIVERY METHODS

Nanoparticle Complexing.

Polymer and ψmRNA solutions are mixed to form complexes. Variousformulation conditions are tested and optimized: (1) sub-22 nmpolyethylenimine (PEI)/mRNA complexes are made by addition of 25 volumesof mRNA to 1 volume of PEI in water with no mixing for 15 minutes. (2)The rod-like poly-L-lysine-polyethylene glycol (PLL-PEG) with averagedimensions of 12×150 nm is synthesized by slow addition of 9 volumes ofmRNA to 1 volume of CK₃₀-PEG_(10k) in acetate counterion buffer whilevortexing. (3) For synthesis of biodegradable gene carrier polymer,polyaspartic anhydride co-ethylene glycol (PAE) is synthesized by ringopening polycondensation of N-(B enzyloxycarbonyl)L-aspartic anhydrideand ethylene glycol. Then, the pendent amine of aspartic acid isdeprotected and protonated by acidification with hydrogen chloride andcondensed with mRNA. (4) For latest generation of nanoparticles, aliquotstock CK₃₀PEG_(10k) as ammonium acetate (1.25 mL; 6.4 mg/mL) is added tosiliconized Eppendorf tubes. Then mRNA is added slowly to CK₃₀PEG_(10k)(2.5 mg in 11.25 mL RNase free H₂O) over 1-2 mins. After 15 mins, it isdiluted 1:2 in RNase-free H₂O.

Intratracheal Delivery.

Mice are anesthetized with 3% halothane (70% N₂O+30% O₂) in ananesthetic chamber and maintained with 1% halothane (70% N₂O+30% O₂)during operation using a nose cone. Trachea os exposed, and 50 μl ofmRNA complex is infused with 150 μl air into the lung through thetrachea using 250 μl Hamilton syringe (Hamilton, Reno, Nev.) with a 27 G½″ needle.

Results

To improve efficiency of delivery and expression of ψmRNA administeredvia the intratracheal (i.t.) route, ψmRNA is encapsulated innanoparticles. Nanoparticle packaging involves condensing andencapsulating DNA (for example) into particles that are smaller than thepore of the nuclear membrane, using chemicals including poly-L-lysineand polyethylene glycol. RNA is packaged into 4 different nanoparticleformulations (PEI, PLL, PAE, and CK₃₀PEG_(10k)), and efficiency of ψmRNAdelivery is compared for luciferase-encoding ψmRNA compare the(Luc-ψmRNA). Delivery kinetics and dose response are then characterizedusing EPO-ψmRNA.

Example 25 Prevention of Restenosis by Delivery to the Carotid Artery ofRecombinant Heat Shock Protein-Encoding, Modified mRNA

Materials and Experimental Methods

Experimental Design

RNA is administered to the carotid artery of rats by intra-arterialinjection near the time of balloon angioplasty, after which blood flowis reinstated. Rats are sacrificed 3 h following injection, carotidartery sections are excised, vascular endothelial cells are harvestedand homogenized, and luciferase activity is determined as described inabove Examples.

Results

Luciferase-encoding pseudouridine-modified RNA is administered to ratcarotid arteries. 3 hours later, luciferase RNA can be detected at thedelivery site but not the adjacent sites.

Next, this protocol is used to prevent restenosis of a blood vesselfollowing balloon angioplasty in an animal restenosis model, by deliveryof modified RNA encoding a heat shock protein, e.g. HSP70; a growthfactor (e.g. platelet-derived growth factor (PDGF), vascular endothelialgrowth factor (V-EGF), or insulin-like growth factor (IGF); or a proteinthat down-regulates or antagonizes growth factor signaling.Administration of modified RNA reduces incidence of restenosis.

Example 26 Treatment of Cystic Fibrosis by Delivery of CFTR-EncodingModified mRNA Molecules to Respiratory Epithelium

CFTR-encoding pseudouridine- or nucleoside-modified RNA is delivered, asdescribed in Example 16, to the lungs of a cystic fibrosis animal model,and its effect on the disease is assessed as described in Scholte B J,et al (Animal models of cystic fibrosis. J Cyst Fibros 2004; 3 Supp12:183-90) or Copreni E, et al, Lentivirus-mediated gene transfer to therespiratory epithelium: a promising approach to gene therapy of cysticfibrosis. GeneTher 2004; 11 Suppl 1: S67-75). Administration of the RNAameliorates cystic fibrosis.

In additional experiments, modified mRNA molecules of the presentinvention are used to deliver to the lungs, other recombinant proteinsof therapeutic value, e.g. via an inhaler that delivers RNA.

Example 27 Treatment of XLA by Delivery of ADA-Encoding Modified mRNAMolecules to Hematopoietic Cells

ADA-encoding pseudouridine- or nucleoside-modified RNA is delivered tothe hematopoietic cells of an X-linked agammaglobulinemia animal model,and its effect on the disease is assessed as described in Tanaka M,Gunawan F, et al, Inhibition of heart transplant injury and graftcoronary artery disease after prolonged organ ischemia by selectiveprotein kinase C regulators. J Thorac Cardiovasc Surg 2005; 129(5):1160-7) or Zonta S, Lovisetto F, et al, Uretero-neocystostomy in a swinemodel of kidney transplantation: a new technique. J Surg Res. 2005April; 124(2):250-5). Administration of the RNA is found to improve XLA.

Example 28 Prevention of Organ Rejection by Delivery ofImmuno-Modulatory Protein-Encoding Modified mRNA Molecules to ATransplant Site

Pseudouridine- or nucleoside-modified RNA encoding a cytokine, achemokine, or an interferon IS (e.g. IL-4, IL-13, IL-I0, or TGF-β) isdelivered to the transplant site of an organ transplant rejection animalmodel, and its effect on the incidence of rejection is assessed asdescribed in Yu P W, Tabuchi R S et al, Sustained correction of B-celldevelopment and function in a murine model of X-linkedagammaglobulinemia (XLA) using retroviral-mediated gene transfer. Blood.2004 104(5): 1281-90) or Satoh M, Mizutani A et al, X-linkedimmunodeficient mice spontaneously produce lupus-related anti20 RNAhelicase A autoantibodies, but are resistant to pristane-induced lupus.Int Immunol 2003, 15(9):1117-24). Administration of the RNA reducesincidence of transplant rejection.

Example 29 Treatment of Niemann-Pick Disease, Mucopolysaccharidosis, andOther Inborn Metabolic Errors by Delivery of Modified mRNA to BodyTissues

Sphingomyelinase-encoding pseudouridine- or nucleoside-modified RNA isdelivered to the lung, brain, or other tissue of Niemann-Pick diseaseType A and B animal models, and its effect on the disease is assessed asdescribed in Passini M A, Macauley S L, et al, AAV vector-mediatedcorrection of brain pathology in a mouse model of Niemann-Pick Adisease. Mol Ther 2005; 11(5): 754-62) or Buccoliero R, Ginzburg L, etal, Elevation of lung surfactant phosphatidylcholine in mouse models ofSandhoff and of Niemann-Pick A disease. J Inherit Metab Dis 2004; 27(5):641-8). Administration of the RNA is found to improve the disease.

Pseudouridine- or nucleoside-modified RNA encoding alpha-L-iduronidase,iduronate-2-sulfatase, or a related enzyme is delivered to the bodytissues of a mucopolysaccharidosis animal model of, and its effect onthe disease is assessed as described in Simonaro C M, D'Angelo M, et al,Joint and bone disease in mucopolysaccharidoses VI and VII:identification of new therapeutic targets and biomarkers using animalmodels. Pediatr Res 2005; 57(5 Pt 1): 701-7) or McGlynn R, Dobrenis K,et al, Differential subcellular localization of cholesterol,gangliosides, and glycosaminoglycans in murine models ofmucopolysaccharide storage disorders. J Comp Neurol 2004 20; 480(4):415-26). Administration of the RNA ameliorates the disease.

In additional experiments, modified mRNA molecules of the presentinvention are used to provide clotting factors (e.g. for hemophiliacs).In additional experiments, modified mRNA molecules of the presentinvention are used to provide acid-b-glucosidase for treating Gaucher's.In additional experiments, modified mRNA molecules of the presentinvention are used to provide alpha-galactosidase A for treating Fabry'sdiseases. In additional experiments, modified mRNA molecules of thepresent invention are used to provide cytokines for treatment ofinfectious diseases.

In additional experiments, modified mRNA molecules of the presentinvention are used to correct other inborn errors of metabolism, byadministration of mRNA molecules encoding, e.g. ABCA4; ABCD3; ACADM;AGL; AGT; ALDH4A1; ALPL; AMPD1; APOA2; AVSD1; BRCD2; C1QA; C1QB; C1QG;C8A; C8B; CACNA1S; CCV; CD3Z; CDC2L1; CHML; CHS1; CIAS1; CLCNKB; CMD1A;CMH2; CMM; COL11AI; COL8A2; COL9A2; CPT2; CRB1; CSE; CSF3R; CTPA; CTSK;DBT; DIO1; DISC1; DPYD; EKV; ENO1; ENO1P; EPB41; EPHX1; F13B; F5;FCGR2A; FCGR2B; FCGR3A; FCHL; FH; FMO3; FMO4; FUCA1; FY; GALE; GBA;GFND; GJA8; GJB3; GLC3B; HF1; HMGCL; HPC1; HRD; HRPT2; HSD3B2; HSPG2;KCNQ4; KCS; KIF1B; LAMB3; LAMC2; LGMD1B; LMNA; LOR; MCKD1; MCL1; MPZ;MTHFR; MTR; MUTYH; MYOC; NB; NCF2; NEM1; NPHS2; NPPA; NRAS; NTRK1;OPTA2; PBX1; PCHC; PGD; PHA2A; PHGDH; PKLR; PKP1; PLA2G2A; PLOD; PPDX;PPT1; PRCC; PRG4; PSEN2; PTOS1; REN; RFX5; RHD; RMD1; RPE65; SCCD;SERPINC1; SJS1; SLC19A2; SLC2A1; SPG23; SPTA1; TAL1; TNFSF6; TNNT2;TPM3; TSHB; UMPK; UOX; UROD; USH2A; VMGLOM; VWS; WS2B; ABCB11; ABCG5;ABCG8; ACADL; ACP1; AGXT; AHHR; ALMS1; ALPP; ALS2; APOB; BDE; BDMR; BJS;BMPR2; CHRNA1; CMCWTD; CNGA3; COL3A1; COL4A3; COL4A4; COL6A3; CPS1;CRYGA; CRYGEP1; CYP1B1; CYP27A1; DBI; DES; DYSF; EDAR; EFEMP1; EIF2AK3;ERCC3; FSHR; GINGF; GLC1B; GPD2; GYPC; HADHA; HADHB; HOXD13; HPE2; IGKC;IHH; IRS1; ITGA6; KHK; KYNU; LCT; LHCGR; LSFC; MSH2; MSH6; NEB; NMTC;NPHP1; PAFAH1P1; PAX3; PAX8; PMS1; PNKD; PPH1; PROC; REGIA; SAG; SFTPB;SLC11A1; SLC3A1; SOS1; SPG4; SRD5A2; TCL4; TGFA; TMD; TPO; UGT1A@; UV24;WSS; XDH; ZAP70; ZFHX1B; ACAA1; AGS1; AGTR1; AHSG; AMT; ARMET; BBS3;BCHE; BCPM; BTD; CASR; CCR2; CCR5; CDL1; CMT2B; COL7A1; CP; CPO; CRY;CTNNB1; DEM; ETM1; FANCD2; F1H; FOXL2; GBE1; GLB1; GLC1C; GNAI2; GNAT1;GP9; GPX1; HGD; HRG; ITIH1; KNG; LPP; LRS1; MCCC1; MDS1; MHS4; MITF;MLH1; MYL3; MYMY; OPA1; P2RY12; PBXPI; PCCB; POU1FI; PPARG; PROS1;PTHR1; RCA1; RHO; SCAT; SCLC1; SCN5A; SI; SLC25A20; SLC2A2; TF; TGFBR2;THPO; THRB; TKT; TM4SF1; TRH; UMPS; UQCRC1; USH3A; VHL; WS2A; XPC;ZNF35; ADH1B; ADH1C; AFP; AGA; AIH2; ALB; ASMD; BFHD; CNGA1; CRBM; DCK;DSPP; DTDP2; ELONG; ENAM; ETFDH; EVC; F11; FABP2; FGA; FGB; FGFR3; FGG;FSHMD1A; GC; GNPTA; GNRHR; GYPA; HCA; HCL2; HD; HTN3; HVBS6; IDUA; IF;JPD; KIT; KLKB1; LQT4; MANBA; MLLT2; MSX1; MTP; NR3C2; PBT; PDE6B; PEE1;PITX2; PKD2; QDPR; SGCB; SLC25A4; SNCA; SOD3; STATH; TAPVR1; TYS; WBS2;WFS1; WHCR; ADAMTS2; ADRB2; AMCN; AP3BI; APC; ARSB; B4GALT7; BHR1; C6;C7; CCAL2; CKN1; CMDJ; CRHBP; CSF1R; DHFR; DIAPH1; DTR; EOS; EPD; ERVR;F12; FBN2; GDNF; GHR; GLRA1; GM2A; HEXB; HSD17B4; ITGA2; KFS; LGMD1A;LOX; LTC4S; MAN2A1; MCC; MCCC2; MSH3; MSX2; NR3C1; PCSK1; PDE6A; PFBI;RASA1; SCZD1; SDHA; SGCD; SLC22A5; SLC26A2; SLC6A3; SM1; SMA@; SMN1;SMN2; SPINK5; TCOF1; TELAB1; TGFBI; ALDH5A1; ARG1; AS; ASSP2; BCKDHB;BF; C2; C4A; CDKN1A; COL10A1; COL11A2; CYP21A2; DYX2; EJM1; ELOVL4;EPM2A; ESR1; EYA4; F13A1; FANCE; GCLC; GJA1; GLYS1; GMPR; GSE; HCR; HFE;HLA-A; HLA-DPB1; HLA-DRA; HPFH; ICS1; IDDM1; IFNGR1; IGAD1; IGF2R; ISCW;LAMA2; LAP; LCA5; LPA; MCDR1; MOCS1; MUT; MYB; NEU1; NKS1; NYS2; OA3;OODD; OFC1; PARK2; PBCA; PBCRA1; PDB1; PEX3; PEX6; PEX7; PKHD1; PLA2G7;PLG; POLH; PPAC; PSORS1; PUJO; RCD1; RDS; RHAG; RP14; RUNX2; RWS; SCA1;SCZD3; SIASD; SOD2; ST8; TAP1; TAP2; TFAP2B; TNDM; TNF; TPBG; TPMT;TULP1; WISP3; AASS; ABCB1; ABCB4; ACHE; AQP1; ASL; ASNS; AUTS1; BPGM;BRAF; C7orf2; CACNA2D1; CCM1; CD36; CFTR; CHORDOMA; CLCN1; CMH6; CMT2D;COL1A2; CRS; CYMD; DFNA5; DLD; DYT11; EEC1; ELN; ETV1; FKBP6; GCK;GNRHR; GHS; GLI3; GPDS1; GUSB; HLXB9; HOXA13; HPFH2; HRX; IAB; IMMP2L;KCNH2; LAMB1; LEP; MET; NCF1; NM; OGDH; OPN1SW; PEX1; PGAM2; PMS2; PON1;PPP1R3A; PRSS1; PTC; PTPN12; RP10; RP9; SERPINE1; SGCE; SHFM1; SHH;SLC26A3; SLC26A4; SLOS; SMAD1; TBXAS1; TWIST; ZWS1; ACHM3; ADRB3; ANKI;CA1; CA2; CCAL1; CLN8; CMT4A; CNGB3; COH1; CPP; CRH; CYP11B1; CYP11B2;DECR1; DPYS; DURS1; EBS1; ECA1; EGI; EXT1; EYA1; FGFR1; GNRH1; GSR;GULOP; HR; KCNQ3; KFM; KWE; LGCR; LPL; MCPH1; MOS; MYC; NAT1; NAT2;NBS1; PLAT; PLEC1; PRKDC; PXMP3; RP1; SCZD6; SFTPC; SGM1; SPG5A; STAR;TG; TRPS1; TTPA; VMD1; WRN; ABCA1; ABL1; ABO; ADAMTS13; AK1; ALAD;ALDH1A1; ALDOB; AMBP; AMCD1; ASS; BDMF; BSCL; C5; CDKN2A; CHAC; CLA1;CMD1B; COL5A1; CRAT; DBH; DNAI1; DYS; DYT1; ENG; FANCC; FBP1; FCMD;FRDA; GALT; GLDC; GNE; GSM1; GSN; HSD17B3; HSN1; IBM2; INVS; JBTS1;LALL; LCCS1; LCCS; LGMD2H; LMX1B; MLLT3; MROS; MSSE; NOTCH1; ORM1;PAPPA; PIP5K1B; PTCH; PTGS1; RLN1; RLN2; RMRP; ROR2; RPD1; SARDH;SPTLC1; STOM; TDFA; TEK; TMC1; TRIM32; TSC1; TYRP1; XPA; CACNB2;COL17A1; CUBN; CXCL12; CYP17; CYP2C19; CYP2C9; EGR2; EMX2; ERCC6; FGFR2;HK1; HPSI; IL2RA; LGI1; LIPA; MAT1A; MBL2; MKI67; MXI1; NODAL; OAT;OATL3; PAX2; PCBD; PEO1; PHYH; PNL1P; PSAP; PTEN; RBP4; RDPA; RET;SFTPA1; SFTPD; SHFM3; SIAL; THC2; TLX1; TNFRSF6; UFS; UROS; AA; ABCC8;ACAT1; ALX4; AMPD3; ANC; APOA1; APOA4; APOC3; ATM; BSCL2; BWS; CALCA;CAT; CCND1; CD3E; CD3G; CD59; CDKN1C; CLN2; CNTF; CPT1A; CTSC; DDB1;DDB2; DHCR7; DLAT; DRD4; ECB2; ED4; EVR1; EXT2; F2; FSHB; FTH1; G6PT1;G6PT2; GIF; HBB; HBBP1; HBD; HBE1; HBG1; HBG2; HMBS; HND; HOMG2; HRAS;HVBS1; IDDM2; IGER; INS; JBS; KCNJ11; KCNJ1; KCNQ1; LDHA; LRP5; MEN1;MLL; MYBPC3; MYO7A; NNO1; OPPG; OPTB1; PAX6; PC; PDX1; PGL2; PGR; PORC;PTH; PTS; PVRL1; PYGM; RAG1; RAG2; ROM1; RRAS2; SAA1; SCA5; SCZD2; SDHD;SERPING1; SMPD1; TCIRG1; TCL2; TECTA; TH; TREH; TSG101; TYR; USH1C;VMD2; VRN1; WT1; WT2; ZNF145; A2M; AAAS; ACADS; ACLS; ACVRL1; ALDH2;AMHR2; AOM; AQP2; ATD; ATP2A2; BDC; C1R; CD4; CDK4; CNA1; COL2A1;CYP27B1; DRPLA; ENUR2; FEOM1; FGF23; FPF; GNB3; GNS; HAL; HBP1; HMGA2;HMN2; HPD; IGF1; KCNA1; KERA; KRAS2; KRT1; KRT2A; KRT3; KRT4; KRT5;KRT6A; KRT6B; KRTHB6; LDHB; LYZ; MGCT; MPE; MVK; MYL2; OAP; PAH; PPKB;PRB3; PTPN11; PXR1; RLS; RSN; SAS; SAX1; SCA2; SCNN1A; SMAL; SPPM;SPSMA; TBX3; TBX5; TCF1; TPI1; TSC3; ULR; VDR; VWF; ATP7B; BRCA2; BRCD1;CLN5; CPB2; ED2; EDNRB; ENUR1; ERCC5; F10; F7; GJB2; GJB6; IPF1; MBS1;MCOR; NYS4; PCCA; RB1; RHOK; SCZD7; SGCG; SLC10A2; SLC25A15; STARP1;ZNF198; ACHM1; ARVDI; BCH; CTAA1; DAD1; DFNB5; EML1; GALC; GCH1; IBGC1;IGH@; IGHC group; IGHG1; IGHM; IGHR; IV; LTBP2; MCOP; MJD; MNG1 MPD1;MPS3C; MYH6; MYH7; NP; NPC2; PABN1; PSEN1 PYGL; RPGRIP1; SERPINA1;SERPINA3; SERPINA6; SLC7A7; SPG3A; SPTB; TCL1A; TGMI; TITF1; TMIP; TRA@;TSHR; USH1A; VP; ACCPN; AHO2; ANCR; B2M; BBS4; BLM; CAPN3; CDAN1; CDAN3;CLN6; CMH3; CYP19; CYP1A1; CYP1A2; DYX1; EPB42; ETFA; EYCL3; FAH; FBN1;FES; HCVS; HEXA; IVD; LCS1; LIPC; MYO5A; OCA2; OTSC1; PWCR; RLBP1;SLC12A1; SPG6; TPM1; UBE3A; WMS; ABCC6; ALDOA; APRT; ATP2A1; BBS2;CARD15; CATM; CDH1; CETP; CHST6; CLN3; CREBBP; CTH; CTM; CYBA; CYLD;DHS; DNASE1; DPEP1; ERCC4; FANCA; GALNS; GAN; HAGH; HBA1; HBA2; HBHR;HBQ1; HBZ; HBZP; HP; HSD11B2; IL4R; LIPB; MC2R; MEFV; MHC2TA; MLYCD;MMVP1; PHKB; PHKG2; PKD1; PKDTS; PMM2; PXE; SALL1; SCA4; SCNN1B; SCNN1G;SLC12A3; TAT; TSC2; VDI; WT3; ABR; ACACA; ACADVL; ACE; ALDH3A2; APOH;ASPA; AXIN2; BCL5; BHD; BLMH; BRCA1; CACD; CCA1; CCZS; CHRNB1; CHRNE;CMT1A; COL1A1; CORD5; CTNS; EPX; ERBB2; G6PC; GAA; GALK1; GCGR; GFAP;GH1; GH2; GP1BA; GPSC; GUCY2D; ITGA2B; ITGB3; ITGB4; KRT10; KRT12;KRT13; KRT14; KRT14L1; KRT14L2; KRT14L3; KRT16; KRT16L1; KRT16L2; KRT17;KRT9; MAPT; MDB; MDCR; MGI; MHS2; MKS1; MPO; MYO15A; NAGLU; NAPB; NF1;NME1; P4HB; PAFAH1B1; PECAM1; PEX12; PHB; PMP22; PRKAR1A; PRKCA;PRKWNK4; PRP8; PRPF8; PTLAH; RARA; RCV1; RMSA1; RP17; RSS; SCN4A;SERPINF2; SGCA; SGSH; SHBG; SLC2A4; SLC4A1; SLC6A4; SMCR; SOST; SOX9;SSTR2; SYM1; SYNS1; TCF2; THRA; TIMP2; TOC; TOP2A; TP53; TRIM37; VBCH;ATP8B1; BCL2; CNSN; CORD1; CYB5; DCC; F5F8D; FECH; PEO; LAMA3; LCFS2;MADH4; MAFD1; MC2R; MCL; MYP2; NPC1; SPPK; TGFBRE; TGIF; TTR; AD2; AMH;APOC2; APOE; ATHS; BAX; BCKDHA; BCL3; BFIC; C3; CACNA1A; CCO; CEACAM5;COMP; CRX; DBA; DDU; DFNA4; DLL3; DM1; DMWD; E11S; ELA2; EPOR; ERCC2;ETFB; EXT3; EYCL1; FTL; FUT1; FUT2; FUT6; GAMT; GCDH; GPI; GUSM; HB1;HCL1; HHC2; HHC3; ICAM3; INSR; JAK3; KLK3; LDLR; LHB; LIG1; LOH19CR1;LYL1; MAN2B1; MCOLN1; MDRV; MLLT1; NOTCH3; NPHS1; OFC3; OPA3; PEPD;PRPF31; PRTN3; PRX; PSG1; PVR; RYR1; SLC5A5; SLC7A9; STK11; TBXA2R;TGFB1; TNNI3; TYROBP; ADA; AHCY; AVP; CDAN2; CDPD1; CHED1; CHED2;CHRNA4; CST3; EDN3; EEGV1; FTLL1; GDF5; GNAS; GSS; HNF4A; JAG1; KCNQ2;MKKS; NBIA1; PCK1; PI3; PPCD; PPGB; PRNP; THBD; TOP1; AIRE; APP; CBS;COL6A1; COL6A2; CSTB; DCR; DSCR1; FPDMM; HLCS; HPE1; ITGB2; KCNE1; KNO;PRSS7; RUNX1; SOD1; TAM; ADSL; ARSA; BCR; CECR; CHEK2; COMT; CRYBB2;CSF2RB; CTHM; CYP2D6; CYP2D7P1; DGCR; DIA1; EWSR1; GGT1; MGCR; MN1;NAGA; NF2; OGS2; PDGFB; PPARA; PRODH; SCO2; SCZD4; SERPIND1; SLC5A1;SOX10; TCN2; TIMP3; TST; VCF; ABCD1; ACTL1; ADFN; AGMX2; AHDS; AIC;AIED; AIH3; ALAS2; AMCD; AMELX; ANOP1; AR; ARAF1; ARSC2; ARSE; ARTS;ARX; ASAT; ASSP5; ATP7A; ATRX; AVPR2; BFLS; BGN; BTK; BZX; C1HR;CACNA1F; CALB3; CBBM; CCT; CDR1; CFNS; CGF1; CHM; CHR39C; CIDX; CLA2;CLCN5; CLS; CMTX2; CMTX3; CND; COD1; COD2; COL4A5; COL4A6; CPX; CVD1;CYBB; DCX; DFN2; DFN4; DFN6; DHOF; DIAPH2; DKC1; DMD; DSS; DYT3; EBM;EBP; ED1; ELK1; EMD; EVR2; F8; F9; FCP1; FDPSL5; FGD1; FGS1; FMR1; FMR2;G6PD; GABRA3; GATA1; GDI1; GDXY; GJB1; GK; GLA; GPC3; GRPR; GTD; GUST;HMS1; HPRT1; HPT; HTC2; HTR2C; HYR; IDS; IHG1; IL2RG; INDX; IP1; IP2;JMS; KAL1; KFSD; L1CAM; LAMP2; MAA; MAFD2; MAOA; MAOB; MCF2; MCS; MEAX;MECP2; MF4; MGC1; MIC5; MID1; MLLT7; MLS; MRSD; MRX14; MRX1; MRX20;MRX2; MRX3; MRX40; MRXA; MSD; MTM1; MYCL2; MYP1; NDP; NHS; NPHL1; NR0B1;NSX; NYS1; NYX; OA1; OASD; OCRL; ODT1; OFD1; OPA2; OPD1; OPEM; OPN1LW;OPN1MW; OTC; P3; PDHA1; PDR; PFC; PFKFB1; PGK1; PGK1P1; PGS; PHEX;PHKA1; PHKA2; PHP; PIGA; PLP1; POF1; POLA; POU3F4; PPMX; PRD; PRPS1;PRPS2; PRS; RCCP2; RENBP; RENS1; RP2; RP6; RPGR; RPS4X; RPS6KA3; RS1;S11; SDYS; SEDL; SERPINA7; SH2D1A; SHFM2; SLC25A5; SMAX2; SRPX; SRS;STS; SYN1; SYP; TAF1; TAZ; TBX22; TDD; TFE3; THAS; THC; TIMM8A; TIMP1;TKCR; TNFSF5; UBE1; UBE2A; WAS; WSN; WTS; WWS; XIC; XIST; XK; XM; XS;ZFX; ZIC3; ZNF261; ZNF41; ZNF6; AMELY; ASSP6; AZF1; AZF2; DAZ; GCY;RPS4Y; SMCY; SRY; ZFY; ABAT; AEZ; AFA; AFD1; ASAH1; ASD1; ASMT; CCAT;CECR9; CEPA; CLA3; CLN4; CSF2RA; CTS1; DF; DIH1; DWS; DYT2; DYT4; EBR3;ECT; EEF1A1L14; EYCL2; FANCB; GCSH; GCSL; GIP; GTS; HHG; HMI; HOAC;HOKPP2; HRPT1; HSD3B3; HTC1; HV1S; ICHQ; ICR1; ICR5; IL3RA; KAL2; KMS;KRT18; KSS; LCAT; LHON; LIMM; MANBB; MCPH2; MEB; MELAS; MIC2; MPFD; MS;MSS; MTATP6; MTCO1; MTCO3; MTCYB; MTND1; MTND2; MTND4; MTND5; MTND6;MTRNR1; MTRNR2; MTTE; MTTG; MTTI; MTTK; MTTL1; MTTL2; MTTN; MTTP; MTTS1;NAMSD; OCD1; OPD2; PCK2; PCLD; PCOS1; PFKM; PKD3; PRCA1; PRO1; PROP1;RBS; RFXAP; RP; SHOX; SLC25A6; SPG5B; STO; SUOX; THM; or TTD.

Example 30 Treatment of Vasospasm by Delivery of iNOS-Encoding ModifiedmRNA MOLECULES TO BODY TISSUES

Inducible nitric oxide synthase (iNOS)-encoding pseudouridine- ornucleoside-modified RNA is delivered to the vascular endothelium ofvasospasm animals models (e.g. subarachnoid hemorrhage), and its effecton the disease is assessed as described in Pradilla G, Wang P P, et al,Prevention of vasospasm by anti-CD11/CD18 monoclonal antibody therapyfollowing subarachnoid hemorrhage in rabbits. J Neurosurg 2004; 101(1):88-92) or Park S, Yamaguchi M, et al, Neurovascular protection reducesearly brain injury after subarachnoid hemorrhage. Stroke 2004; 35(10):2412-7). Administration of the RNA ameliorates the disease.

Example 31 Restoration of Hair Growth by Delivery of Modified mRNAEncoding an Immunosuppressive Protein

Pseudouridine- or nucleoside-modified RNA encoding a telomerase or animmunosuppressive protein (e.g. α-MSH, TGF-β 1, or IGF-I is delivered tohair follicles of animals used as models of hair loss or balding, andits effect on hair growth is assessed as described in Jiang J, Tsuboi R,et al, Topical application of ketoconazole stimulates hair growth inC3H/HeN mice. J Dermatol 2005; 32(4): 243-7) or McElwee K J,Freyschmidt-Paul P, et al, Transfer of CD8(+) cells induces localizedhair loss whereas CD4(+)/CD25(−) cells promote systemic alopecia areataand CD4(+)/CD25(+) cells blockade disease onset in the C3H/HeJ mousemodel. J Invest Dermatol 2005; 124(5): 947-57). Administration of theRNA restores hair growth.

Example 32 Synthesis of an In Vitro-Transcribed RNA Molecule withAltered Nucleosides Containing an siRNA

A double-stranded RNA (dsRNA) molecule comprising pseudouridine or amodified nucleoside and further comprising a small interfering RNA(siRNA) or short hairpin RNA (shRNA) is synthesized by the followingprocedure: Complementary RNA strands with the desired sequencecontaining uridine or 1 or more modified nucleosides are synthesized byin vitro transcription (e.g. by T7, SP6, or T3 phage RNA polymerase) asdescribed in Example 5. dsRNA molecules exhibit reduced immunogenicity.In other experiments, the dsRNA molecules are designed to be processedby a cellular enzyme to yield the desired siRNA or shRNA. Because dsRNAmolecules of several hundred nucleotides are easily synthesized, eachdsRNA may also be designed to contain several siRNA or shRNA molecules,to facilitate delivery of multiple siRNA or shRNA to a single targetcell.

Example 33 Use of an In Vitro-Transcribed RNA Molecule with AlteredNucleosides to Deliver siRNA

The dsRNA molecule of the previous Example is complexed with atransfection reagent (e.g a cationic transfection reagent, a lipid-basedtransfection reagent, a protein-based transfection reagent, apolyethyleneimine based transfection reagent, or calcium phosphate) anddelivered to a target cell of interest. Enzymes in or on the surface ofthe target cell degrade the dsRNA to the desired siRNA or shRNAmolecule(s). This method effectively silences transcription of 1 or morecellular genes corresponding to the siRNA or shRNA sequence(s).

Example 34 Testing the Effect of Additional Nucleoside Modifications onRNA Immunogenicity and Efficiency of Translation

Additional nucleoside modifications are introduced into invitro-transcribed RNA, using the methods described above in Examples 5and 10, and their effects on immunogenicity translation efficiency aretested as described in Examples 4-11 and 12-18, respectively. Certainadditional modifications are found to decrease immunogenicity andenhance translation. These modifications are additional embodiments ofmethods and compositions of the present invention.

Modifications tested include, e.g.:

m¹A; m²A; Am; ms²m⁶A; i⁶A; ms²i⁶A; io⁶A; ms²i0⁶A; g⁶A; t⁶A; ms²t⁶A;m⁶t⁶A; hn⁶A; ms²hn⁶A; Ar(p); I; m¹I; m¹Im; m³C; Cm; S²C; ac⁴C; f⁵c;m⁵Cm; ac⁴Cm; k²c; m¹G; m²G; m⁷G; Gm; m² ₂G; m²Gm; m² ₂Gm; Gr(p); yW;o₂yW; OHyW; OHyW*; imG; mimG; Q; oQ; galQ; manQ; preQ₀; preQ₁; G⁺; D;m⁵Um′, m¹ψ; ψm; S⁴U; m⁵s²U′, S²Um, ′acp³U⋅, ho⁵u., mo⁵U⋅; cmo⁵U⋅;mcmo⁵U; chm⁵U; mchm⁵U⋅; mcm⁵U; mcm⁵Um; mcm⁵s²U; nm⁵s²U; mnm⁵U; mnm⁵s²U;mnm⁵se²U; ncm⁵U; ncm⁵Um; cmnm⁵U; cmnm⁵Um; cmnm⁵s²U; m⁶ ₂A; Im; m⁴C;m⁴Cm; hm⁵C; m³U; m¹acp3ψ; cm⁵U; m⁶Am; m⁶ ₂Am; m^(2,7)G; m^(2,2,7)G;m³Um; m⁵D; m³ψ; f⁵Cm; m¹Gm; m¹Am; τm⁵U; τm⁵s²U; imG-14; imG2; and ac⁶A.

Materials and Methods for Examples 35-38

HPLC Purification of RNA:

mRNA produced by T7 polymerase transcription was purified by HPLC usinga column matrix of alkylated nonporous polystyrene-divinylbenzene(PS-DVB) copolymer microspheres (2.1 μm) (21 mm×100 mm column) and abuffer system of Triethylammonium acetate (TEAA) with an acetonitrilegradient. Buffer A contained 0.1 M TEAA and Buffer B contained 0.1 MTEAA and 25% acetonitrile. Columns were equilibrated with 38% Buffer Bin Buffer A, loaded with RNA, and then run with a linear gradient to 55%Buffer B over 30 minutes at 5 ml/minute. Fractions corresponding to thedesired peak were collected. RNA analyses were performed with the samecolumn matrix and buffer system, but using a 7.8 mm×50 mm column at 1.0ml/min and a gradient duration of 25 minutes.

RNA Isolation from Column Fractions:

Collected fractions were combined, and first their RNA content wasconcentrated using Amicon Ultra-15 centrifugal filter units with 30Kmembrane (Millipore). The filter device was filled with 15 ml sample andspin at 4,000×g for 10 min (4° C.) in a Thermo Scientific Sorvall ST16Rcentrifuge using swinging bucket rotor. Under these conditions, ˜98% ofthe solvent volume can be removed. When the collected fractions had avolume over 15 ml, the filter unit was reused by filling up withadditional column fractions and centrifuging again until all of the RNAwas in one tube. To remove the salt and solvent from the concentratedRNA, nuclease free water was added (up to 15 ml) and the filter devicewas spun again. The process of “washing out” was repeated until theconcentration of the acetonitrile was <0.001%. The desalted andsolvent-free sample was removed from the filtering device and the RNAwas recovered by overnight precipitation at −20° C. in NaOAc (0.3 M, pH5.5), isopropanol (1 volume) and glycogen (3 μl). The precipitated RNAwas collected, washed twice with ice-cold 75% ethanol and reconstitutedin water.

dsRNA Dot Blot:

RNA (25-100 ng) was blotted onto a nitrocellulose membrane, allowed todry, blocked with 5% non-fat dried milk in TBS buffer supplemented with0.05% Tween-20 (TBS-T), and incubated with dsRNA-specific mAb J2 or K1(English & Scientific Consulting) for 60 minutes. Membranes were washed6 times with TBS-T and then reacted with HRP-conjugated donkeyanti-mouse antibody (Jackson Immunology). After washing 6 times, dsRNAwas detected with the addition of SuperSignal West Pico Chemiluminescentsubstrate (Pierce) and image capture for 30 seconds to 2 minutes on aFujifilm LAS1000 digital imaging system.

Dendritic Cell Generation:

Monocytopheresis samples were obtained from normal volunteers through anIRB-approved protocol. Human DCs were produced by treating monocyteswith GM-CSF (50 ng/ml)+IL-4 (100 ng/ml) (R&D Systems) in AIM V medium(Invitrogen) for 7 days. On days 3 and 6, a 50% volume of new mediumwith cytokines was added.

Murine DC were generated by isolating bone marrow mononuclear cells fromBalb/c mice and culturing in RPMI+10% FBS medium supplemented withmurine GM-CSF (20 ng/ml, Peprotech). On days 3 and 6, a 50% volume ofnew medium with GM-CSF was added. Non-adherent cells were used after 7days of culture.

Lipofectin Complexing of RNA:

Stock phosphate buffer was added to serum-free DMEM to give finalconcentrations of 20 mM potassium phosphate and 100 ng/ml BSA, pH 6.4.For 3 wells of a 96-well plate, lipofectin complexed RNA was prepared inthe following ratios: 2.4 μl of lipofectin was added to 21.3 μlserum-free DMEM medium with phosphate buffer and incubated at roomtemperature for 10 minutes. Then, 0.75 μg of RNA in 9.9 μl serum-freeDMEM was added and the mixture was incubated for 10 additional minutesat room temperature. Lastly, 116.4 ml serum-free DMEM was added to bringup the final volume to 150 ml. The mixture was vortexed.

TransIT Complexing of RNA:

For each well of a 96-well plate, 0.25 μg of RNA was added to 17.3 μl ofserum-free DMEM on ice. TransIT mRNA reagent (0.3 ul) was added withvortexing followed by 0.2 μl of mRNA boost reagent and vortexing.Complexed RNA was added within 5 minutes of formation.

Cell Transfections:

For lipofectin complexed RNA, 50 μl (0.25 μg RNA/well) was addeddirectly to cells, 5×10⁵ per well. Transfected cells were incubated for1 h at 37° C. in a 5% CO₂ incubator. The lipofectin-RNA mixture wasremoved and replaced with 200 μl pre-warmed serum containing medium. ForTransIT complexed RNA, 17 μl of complex was added to cells, 5×10⁵ perwell, in 200 μl of serum containing medium. Cells were lysed in specificlysis media, 3 to 24 hr after transfection and firefly or renillaluciferase activity was measured with specific substrates in aluminometer.

RNA Immunogenicity Analysis:

DCs (murine or human) in 96-well plates (5×10⁵ cells/well) were treatedwith medium, or lipofectin or TransIT complexed RNA. Supernatant washarvested after 24 hr and subjected to analysis. The levels of IFN-α(TransIT delivered RNA) or TNF-α (Lipofectin delivered RNA) (BiosourceInternational, Camarillo, Calif.) were measured in supernatants byELISA. Cultures were performed in triplicate to quadruplicate andmeasured in duplicate.

Example 35

This example examines the sequence and cell type dependency oftranslation of ψ-, m5C, and ψ/m5C-modified mRNA relative to theunmodified (U) RNA. Firefly or Renilla luciferase encoding mRNA with theindicated modifications were complexed to lipofectin and delivered tomurine dendritic (A) and HEK293T (B) cells. Human DC were transfectedwith firefly or renilla luciferase-encoding mRNA with the indicatedmodifications complexed with TransIT (C). The data demonstrates thatdepending on the sequence of the RNA and the type of cell translatingit, the optimal modification varies. It also shows that the enhancementcaused by incorporation of modified nucleosides is substantially greaterfor primary cells compared to transformed cell lines. Enzyme activity inlysed cells was measured using specific substrates and measurement oflight produced in a luminometer and expressed as fold increase comparedto unmodified (U) RNA.

Example 36

Phage polymerase transcription reactions used for the generation of mRNAresults in large quantities of RNA of the correct size, but alsocontains contaminants. This is visualized by application of RNA to areverse phase HPLC column that separates RNA based on size underdenaturing conditions. Y-modified TEV-luciferase-A51 RNA was applied tothe HPLC column in 38% Buffer B and subjected to a linear gradient ofincreasing Buffer B to 55%. The profile demonstrated both smaller thanexpected and larger than expected contaminants. These results are shownin FIG. 22.

Example 37

HPLC purification increases translation of all types of modified orunmodified RNA, but ψ-modified mRNA is translated best. The results areshown in FIG. 23: (A) EPO encoding mRNA with the indicated modificationsand with or without HPLC purification were delivered to murine DCs andEPO levels in the supernatant were measured 24 hr later. While m5C/Y□modified mRNA had the highest level of translation prior to HPLCpurification, ψ-modified mRNA had the highest translation after HPLCpurification. (B) Human DCs were transfected with renilla encoding mRNAwith the indicated modifications with or without HPLC purification.Similar to murine DCs and EPO mRNA, after HPLC purification, Y-modifiedmRNA had the highest level of translation.

Example 38

ψ, m5C, and ψ/m5C-modified mRNA have low levels of immunogenicity thatis reduced to control levels with HPLC purification. The results areshown in FIG. 24: (A) Human DCs were transfected with RNA complexed toTransIT with the indicated modifications with or without HPLCpurification. IFN-α levels were measured after 24 hr. HPLC purificationincreased the immunogenicity of unmodified RNA, which is dependent ofthe sequence, as other unmodified RNAs had similar levels of IFN-α orreduced levels. ψ-modified RNA had unmeasurable levels of IFN-α similarto control treated DCs. (B) ψ-modified RNA before (−) and after HPLCpurification (P1 and P2) was analyzed for dsRNA using dot blotting witha monoclonal antibody specific for dsRNA (J2). Purification of RNAremoved dsRNA contamination. (C) ψ-modified RNA encoding iPS factors areimmunogenic, which is removed by HPLC purification of the RNA.

Materials and Methods for Examples 39-41

Cell Culture.

Neonatal human epidermal keratinocytes (HEKn) cells (Invitrogen) werecultured in EpiLife Medium supplemented with keratinocyte growthsupplement and Penicillin/Streptomycin (Invitrogen). All cells weregrown at 37° C. and 5% CO₂. The human iPS cells that were induced usingmethods described herein were transferred to hESC-qualified matrigelmatrix (BD Biosciences) coated 6-well plates after transfection.

Constructions of Vectors.

Generally the same as Examples 1-3.

mRNA Production.

Generally the same as for Examples 1-3.

mRNA Purification and Analysis.

In some experimental embodiments, the mRNA was purified by HPLC, columnfractions were collected, and the mRNA fractions were analyzed forpurity an immunogenicity as described in “Materials and Methods forExamples 35-38” and/or as described and shown for FIGS. 22-24. In somepreferred experimental embodiments, purified RNA preparations comprisingor consisting of mRNAs encoding one or more reprogramming factors whichexhibited little or no immunogenicity were used for the experiments forreprogramming human somatic cells to iPS cells.

Reprogramming of Primary Keratinocytes.

HEKn cells were plated at 1×10⁵ cells/well of a 6-well dish in EpiLifemedium and grown overnight. The cells were transfected with equalamounts of each reprogramming factor mRNA (KLF4, LIN28, c-MYC, NANOG,OCT4, and SOX2) or a subset of the factors using TransIT™ mRNAtransfection reagent (MirusBio, Madison, Wis.). Three transfections wereperformed, every other day, with media changes every day. The day afterthe third transfection, the cells were trypsinized and plated in mTeSR1medium (StemCell Technologies) onto matrigel-coated 6-well plates. ThemTeSR cell medium was changed daily. Cells were maintained at 37° C.with 5% CO₂. Plates were screened for morphology changes using aninverted microscope.

HEKn cells were also reprogrammed by a single transfection byelectroporation with equal amounts of each reprogramming factor mRNA.The cells were plated directly onto matrigel-coated plates at a densityof 1×10⁵ cells per 6-well dish or 7.5×10⁵ cells per 10 cm dish in mTeSR1medium which was changed daily.

Immunofluorescence.

Generally the same as Examples 1-3

Quantitative RT-PCR (qPCR)

Cellular RNA was reverse-transcribed using standard methods and an oligod(T)₂₁ primer from equivalent amounts of cellular RNA. Three messageswere amplified using gene-specific primers and real-time PCR using SYBRgreen detection and GAPDH normalization. Expression levels weredetermined in relation to the level of expression in the original HEKncell line, and depicted as changes in cycle threshold (CT) level.

Example 39

This example describes development of a protocol for iPS cell generationfrom somatic keratinocytes. Equal amounts (by weight) of KLF4, c-MYC,OCT4, and SOX2 mRNAs were transfected into HEKn cells three times (onceevery other day) with TransIT™ mRNA Reagent. Medium was changed daily.The day after the third transfection, the cells were plated ontomatrigel-coated dishes and grown in mTeSR1 cell medium. By 11 days afterthe first transfection the reprogrammed cell morphology began to appear(FIG. 28).

Example 40

This example describes the characterization of cells resulting fromtransfection of primary keratinocytes with equal amounts of KLF4, LIN28,c-MYC, NANOG, OCT4, and SOX2 mRNAs. One million HEKn cells wereelectoporated once with 5 micrograms of each mRNA and plated onmatrigel-coated 10 cm dishes in mTeSR1 cell medium. 15 Days aftertransfection the cells were fixed for immunofluorescence analysis. Theresulting colonies were positive for the iPS markers KLF4, LIN28, SSEA4,TRA-1-60, and NANOG (FIG. 29).

Example 41

This example describes expression differences between primarykeratinocytes and keratinocytes reprogrammed with equal amounts of KLF4,c-MYC, OCT4, and SOX2 mRNAs. 7.5×10⁵ HEKn cells were electoporated oncewith 3 or 5 micrograms of each mRNA and plated on matrigel-coated 10 cmdishes in mTeSR1 medium. Medium was changed daily. 13 days aftertransfection the cells were transferred to freshly-coated matrigelplates. 21 days after transfection, total cellular RNA was purified fromuntransfected HEKn cells and two wells of reprogrammed cells. An equalamounts of each cellular RNA was converted to cDNA and analyzed by qPCR.Increased levels of NANOG, CRIPTO and REX1 were detected by qPCR usingmessage-specific primers (FIG. 30). These three messages have been shownto be elevated in iPS cells (Aasen T et al. 2008. Nature Biotech 26:1276). None of these factors was introduced into the cells bytransfection; therefore the changes in expression are due to theinfluence of the reprogramming factors that were introduced.

Example 42 Transdifferentiating Cells with mRNAs

Cells can be transdifferentiated using the purified mRNA preparationsdescribed herein, or the modified mRNAs described herein, or thepurified mRNA preparations containing modified mRNAs described herein.In this Example, a purified RNA preparation containing OCT4 mRNA thathas at least one pseudouridine or one 5-methycytidine is employed. Suchpurified and modified OCT4 mRNAs are substituted for the OCT4 encodingvectors in the protocol described by Szabo et al. (Nature 468: 521-528,2010, which is herein incorporated by reference in its entirety as iffully set forth herein) and in the protocol described in Racila et al.(Gene Therapy, 1-10, 2010, herein incorporated by reference in itsentirety as if fully set forth herein). In one embodiment of each ofthese methods, the purified RNA preparation comprises or consists of OCT4 mRNA, wherein all of the uridine nucleosides are replaced bypseudouridine nucleosides. In one embodiment of each of these methods,the purified RNA preparation comprises or consists of OCT 4 mRNA,wherein all of the cytidine nucleosides are replaced by 5-methylcytidinenucleosides. In one embodiment of each of these methods, the purifiedRNA preparation comprises or consists of OCT 4 mRNA, wherein all of theuridine nucleosides are replaced by pseudouridine nucleosides and all ofthe cytidine nucleosides are replaced by 5-methylcytidine nucleosides.In preferred embodiments, the OCT4 mRNA is purified to be free ofcontaminating RNAs. The Racilla et al. reference describes a system inwhich human keratinocytes were transdifferentiated by being redirectedto an alternative differentiation pathway. In particular, transienttransfection of human skin keratinocytes with the transcription factorOCT4 was employed. After 2 days, these transfected cells displayedexpression of endogenous embryonic genes and showed reduced genomicmethylation. It was shown that such cells could be converted intoneuronal and contractile mesenchymal cell types.

The Szabo et al. reference demonstrated the ability to generateprogenitors and mature cells of the haematopoietic fate directly fromhuman dermal fibroblasts without establishing pluripotency. Inparticular, ectopic expression of OCT4 activated haematopoietictranscription factors, together with specific cytokine treatment,allowed generation of cells expressing the pan-leukocyte marker CD45.These unique fibroblast-derived cells gave rise to granulocytic,monocytic, megakaryocytic and erythroid lineages, and demonstrated invivo engraftment capacity.

Besides the use of OCT4, both of these protocols also employed cytokinesor growth factors, such as transforming growth factor (TGF), PDGF-BB,stem cell factor (SCF), and FMS-like tyrosine kinase 3 ligand (Flt3L).Other growth factors and cytokines could be used, such asgranulocyte-colony stimulating factor (G-CSF), IL-3, IL-6,erythropoietin, basic fibroblast growth factor (bFGF), insulin-likegrowth factor 2 (IGFII), and bone morphogenetic protein 4 (BMP-4). Assuch, in certain embodiments, the Racilla et al. or Szabo et al.protocols are repeated with the substitution of modified OCT4 mRNA(e.g., psuedouridine-modified and/or 5-methycytidine-modified), alongwith the use of the growth factors or cytokines recited above. In someembodiments, the cells are contacted with the cytokine and/or growthfactor proteins that are used. In some other embodiments, the cells arecontacted with modified mRNAs (e.g., modified mRNAs as described in thepresent application, e.g., e.g., psuedouridine-modified and/or5-methycytidine-modified) that encode one or more of the cytokinesand/or growth factors that are used in the transdifferentiationprotocol. It will be clear from this description, that the presentinvention includes contacting a human or animal cell with a purified RNApreparation comprising or consisting of mRNA that encodes areprogramming factor in order to transdifferentiate a cell having afirst state of differentiation or phenotype to a cell having a secondstate of differentiation or phenotype.

REFERENCES

-   Aoi T, Yae K, Nakagawa M, Ichisaka T, Okita K, Takahashi K, Chiba T,    Yamanaka S. 2008. Generation of pluripotent stem cells from adult    mouse liver and stomach cells. Science 321: 699-702.-   Banerjee A K. 1980. 5′-terminal cap structure in eucaryotic    messenger ribonucleic acids. Microbiol Rev 44: 175-205.-   Chan E M, et al. 2009. Live cell imaging distinguishes bona fide    human iPS cells from partially reprogrammed cells. Nat Biotechnol    27: 1033-1037.-   Ebert A D, Yu J, Rose F F, Jr., Mattis V B, Lorson C L, Thomson J A,    Svendsen C N. 2009. Induced pluripotent stem cells from a spinal    muscular atrophy patient. Nature 457: 277-280.-   Edmonds M. 1990. Polyadenylate polymerases. Methods Enzymol 181:    161-170.-   Feng, R et al. 2008. PU.1 and C/EBPa/b convert fibroblasts into    macrophage-like cells. Proc. Natl Acad. Sci. USA 105: 6057-6062.-   Gershon P D. 2000. (A)-tail of two polymerase structures. Nat Struct    Biol 7: 819-821.-   Gonzalez F, Barragan Monasterio M, Tiscornia G, Montserrat Pulido N,    Vassena R, Batlle Morera L, Rodriguez Piza I, Izpisua Belmonte    J C. 2009. Generation of mouse-induced pluripotent stem cells by    transient expression of a single nonviral polycistronic vector. Proc    Natl Acad Sci USA 106: 8918-8922.-   Graf T, Enver T. 2009. Forcing cells to change lineages. Nature 462:    587-594.-   Grudzien E, Stepinski J, Jankowska-Anyszka M, Stolarski R,    Darzynkiewicz E, Rhoads R E. 2004. Novel cap analogs for in vitro    synthesis of mRNAs with high translational efficiency. RNA 10:    1479-1487.-   Grudzien-Nogalska E, Jemielty J, Kowalska J, Darzynkiewicz E,    Rhoads R. 2007. Phosphorothioate cap analogs stabilize mRNA and    increase translational efficiency in mammalian cells. RNA 13:    1745-1755.-   Higman M A, Bourgeois N, Niles E G. 1992. The vaccinia virus mRNA    (guanine-N7-)-methyltransferase requires both subunits of the mRNA    capping enzyme for activity. J Biol Chem 267: 16430-16437.-   Higman M A, Christen L A, Niles E G. 1994. The mRNA    (guanine-7-)methyltransferase domain of the vaccinia virus mRNA    capping enzyme. Expression in Escherichia coli and structural and    kinetic comparison to the intact capping enzyme. J Biol Chem 269:    14974-14981.-   Huangfu D, Osafune K, Maehr R, Guo W, Eijkelenboom A, Chen S,    Muhlestein W, Melton D A. 2008. Induction of pluripotent stem cells    from primary human fibroblasts with only Oct4 and Sox2. Nat    Biotechnol 26: 1269-1275.-   Ieda, M et al. 2010. Direct reprogramming of fibroblasts into    functional cardiomyocytes by defined factors. Cell 142: 375-386.-   Jemielity J, Fowler T, Zuberek J, Stepinski J, Lewdorowicz M,    Niedzwiecka A, Stolarski R, Darzynkiewicz E, Rhoads R E. 2003. Novel    “anti-reverse” cap analogs with superior translational properties.    RNA 9: 1108-1122.-   Kariko K, Muramatsu H, Welsh F A, Ludwig J, Kato H, Akira S,    Weissman D. 2008. Incorporation of pseudouridine into mRNA yields    superior nonimmunogenic vector with increased translational capacity    and biological stability. Mol Ther 16: 1833-1840.-   Krieg P A, Melton D A. 1984. Functional messenger RNAs are produced    by SP6 in vitro transcription of cloned cDNAs. Nucleic Acids Res 12:    7057-7070.-   Lee G, et al. 2009. Modelling pathogenesis and treatment of familial    dysautonomia using patient-specific iPSCs. Nature 461: 402-406.-   Mackie G A. 1988. Vectors for the synthesis of specific RNAs in    vitro. Biotechnology 10: 253-267.-   Maehr R, Chen S, Snitow M, Ludwig T, Yagasaki L, Goland R, Leibel R    L, Melton D A. 2009. Generation of pluripotent stem cells from    patients with type 1 diabetes. Proc Natl Acad Sci USA 106:    15768-15773.-   Martin S A, Paoletti E, Moss B. 1975. Purification of mRNA    guanylyltransferase and mRNA (guanine-7-) methyltransferase from    vaccinia virions. J Biol Chem 250: 9322-9329.-   Myette J R, Niles E G. 1996. Domain structure of the vaccinia virus    mRNA capping enzyme. Expression in Escherichia coli of a subdomain    possessing the RNA 5′-triphosphatase and guanylyltransferase    activities and a kinetic comparison to the full-size enzyme. J Biol    Chem 271: 11936-11944.-   Nakagawa M, Koyanagi M, Tanabe K, Takahashi K, Ichisaka T, Aoi T,    Okita K, Mochiduki Y, Takizawa N, Yamanaka S. 2008. Generation of    induced pluripotent stem cells without Myc from mouse and human    fibroblasts. Nat Biotechnol 26: 101-106.-   Okita K, Nakagawa M, Hyenjong H, Ichisaka T, Yamanaka S. 2008.    Generation of mouse induced pluripotent stem cells without viral    vectors. Science 322: 949-953.-   Ozawa T, Kishi H, Muraguchi A. 2006. Amplification and analysis of    cDNA generated from a single cell by 5′-RACE: application to    isolation of antibody heavy and light chain variable gene sequences    from single B cells. Biotechniques 40: 469-470, 472, 474 passim.-   Peng Z H, Sharma V, Singleton S F, Gershon P D. 2002. Synthesis and    application of a chain-terminating dinucleotide mRNA cap analog. Org    Lett 4: 161-164.-   Racila D et al. 2010. Transient expression of OCT 4 IS sufficient to    allow human keratinocytes to change their differentiation pathway.    Gene Therapy advance online publication, (Oct. 28, 2010;    doi:10.1038/gt.2010.148).-   Shuman S. 1995. Capping enzyme in eukaryotic mRNA synthesis. Prog    Nucleic Acid Res Mol Biol 50: 101-129.-   Shuman. 2001. Structure, mechanism, and evolution of the mRNA    capping apparatus. Prog Nucleic Acid Res Mol Biol 66: 1-40.-   Shuman S, Surks M, Furneaux H, Hurwitz J. 1980. Purification and    characterization of a GTP-pyrophosphate exchange activity from    vaccinia virions. Association of the GTP-pyrophosphate exchange    activity with vaccinia mRNA guanylyltransferase. RNA    (guanine-7-)methyltransferase complex (capping enzyme). J Biol Chem    255: 11588-11598.-   Stadtfeld M, Nagaya M, Utikal J, Weir G, Hochedlinger K. 2008.    Induced pluripotent stem cells generated without viral integration.    Science 322: 945-949.-   Stepinski J, Waddell C, Stolarski R, Darzynkiewicz E, Rhoads    R E. 2001. Synthesis and properties of mRNAs containing the novel    “anti-reverse” cap analogs 7-methyl(3′-O-methyl)GpppG and 7-methyl    (3′-deoxy)GpppG. RNA 7: 1486-1495.-   Studier F W, Moffatt B A. 1986. Use of bacteriophage T7 RNA    polymerase to direct selective high-level expression of cloned    genes. J Mol Biol 189: 113-130.-   Szabo E, Rampalli S, Risueno R M, Schnerch A, Mitchell R,    Fiebig-Comyn A, Levadoux-Martin M, Bhatia. M. 2010. Direct    conversion of human fibroblasts to multilineage blood progenitors.    Nature. 468: 521-526.-   Takahashi K, Yamanaka S. 2006. Induction of pluripotent stem cells    from mouse embryonic and adult fibroblast cultures by defined    factors. Cell 126: 663-676.-   Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K,    Yamanaka S. 2007. Induction of pluripotent stem cells from adult    human fibroblasts by defined factors. Cell 131: 861-872.-   Vierbuchen T et al. 2010 Direct conversion of fibroblasts to    functional neurons by defined factors. Nature 463: 1035-1041.-   Wang S P, Deng L, Ho C K, Shuman S. 1997. Phylogeny of mRNA capping    enzymes. Proc Natl Acad Sci USA 94: 9573-9578.-   Wilusz J, Shenk T. 1988. A 64 kd nuclear protein binds to RNA    segments that include the AAUAAA polyadenylation motif. Cell 52:    221-228.-   Woltjen K, et al. 2009. piggyBac transposition reprograms    fibroblasts to induced pluripotent stem cells. Nature 458: 766-770.-   Xu C, Inokuma M S, Denham J, Golds K, Kundu P, Gold J D, Carpenter    M K. 2001. Feeder-free growth of undifferentiated human embryonic    stem cells. Nat Biotechnol 19: 971-974.-   Yu J, Hu K, Smuga-Otto K, Tian S, Stewart R, Slukvin, II, Thomson    J A. 2009. Human induced pluripotent stem cells free of vector and    transgene sequences. Science 324: 797-801.-   Yu J, et al. 2007. Induced pluripotent stem cell lines derived from    human somatic cells. Science 318: 1917-1920.-   Zhou H, et al. 2009. Generation of induced pluripotent stem cells    using recombinant proteins. Cell Stem Cell 4: 381-384.

We claim:
 1. A purified RNA preparation comprising in vitro-synthesizedsingle-stranded mRNA molecules that: A) encode at least one iPSCinduction factor selected from the group consisting of OCT4, SOX2, KLF4,LIN28, NANOG, and a MYC protein selected from c-MYC, L-MYC and N-MYC; B)comprise (i) guanosine, (ii) adenosine, (iii) cytidine, (iv)pseudouridine (Ψ) or 1-methyl-pseudouridine (m¹Ψ) in place of uridine,(v) a 5′ cap, and (vi) a 3′ poly(A) tail; and C) have been purifiedusing a purification process that removes RNA contaminant molecules thatare immunogenic and toxic to mammalian cells by inducing an innateimmune response, such that said mRNA molecules lack immunogenicity, ascan be determined using an in vitro monocyte-derived dendritic cell(MDDC) immunogenicity assay by measuring an amount of IFN-α or TNF-αcytokine secreted from human or murine MDDCs transfected with saidpurified RNA preparation that is no greater than the amount of IFN-α orTNF-α secreted by MDDCs transfected with negative controls that do notcontain RNA.
 2. The purified RNA preparation of claim 1, wherein said invitro-synthesized single-stranded mRNA molecules encode 2 or more, 3 ormore, 4 or more, 5 or more, or 6 iPSC induction factors selected fromthe group consisting of OCT4, SOX2, KLF4, LIN28, NANOG, and a MYCprotein selected from c-MYC, L-MYC and N-MYC.
 3. The purified RNApreparation of claim 1 wherein said in vitro-synthesized single-strandedmRNA molecules encode the iPSC induction factors: A) OCT4, SOX2, KLF4and MYC; or B) OCT4, SOX2, KLF4, MYC and LIN28; or C) OCT4, SOX2, KLF4,MYC and NANOG; or D) OCT4, SOX2, KLF4, MYC, LIN28 and NANOG; whereinsaid MYC in A) D) is selected from c-MYC, L-MYC and N-MYC.
 4. Thepurified RNA preparation of claim 1, wherein said purification comprisesat least one purification process selected from the group consisting of:A) HPLC or gravity flow column chromatography; B) treating a compositioncomprising said in vitro-synthesized RNA molecules with one or moreenzymes that specifically digest one or more RNA contaminant moleculesor contaminant DNA molecules; and C) treating a composition comprisingsaid in vitro-synthesized RNA molecules with a ribonuclease III (RNaseIII) enzyme such that short RNase III digestion products are generated,and purifying said short RNase III digestion products away from saidmRNA molecules.
 5. The purified RNA preparation of claim 4, wherein saidpurification further comprises collecting and analyzing fractions fromsaid purification processes for: (i) RNA contaminant molecules, (ii) DNAcontaminant molecules, and (iii) reduced immunogenicity using an invitro MDDC immunogenicity assay, by measuring less secretion of IFN-α orTNF-α from MDDCs transfected with in vitro-synthesized RNA molecules inmore purified fractions.
 6. The purified RNA preparation of claim 4,wherein the result of said purification process is that said invitro-synthesized mRNA molecules can be repeatedly administered withouteliciting an immune response sufficient to eliminate detectableexpression of the recombinant protein or that said in vitro-synthesizedmRNA lacks immunogenicity, enabling repeated delivery without generationof inflammatory cytokines.
 7. The purified RNA preparation of claim 1,wherein at least one of the following applies: (i) greater than 98% ofsaid mRNA molecules comprise a 5′ cap; (ii) said mRNA molecules comprisea cap with a cap0 structure; (iii) said mRNA molecules comprise a capwith a cap1 structure; (iv) all or substantially all said mRNA moleculescomprise a poly(A) tail having 50-200 or greater than 150 nucleotides;(v) said mRNA molecules comprise at least one particular sequenceselected from the group consisting of: a heterologous 5′ UTR sequence, aKozak sequence, an IRES sequence, and a 3′ UTR sequence; (vi) said 5′UTR sequence or 3′ UTR sequence is from a Xenopus or human alpha globinor beta globin mRNA, or wherein said 5′ UTR sequence is a sequence froma tobacco etch virus (TEV) RNA; (vii) said RNA contaminant moleculesthat are removed from said in vitro-synthesized mRNA molecules usingsaid purification process are selected from the group consisting ofdouble-stranded RNA (dsRNA) molecules and un-capped mRNA molecules; or(viii) less than 0.01% of the total RNA in said purified RNA preparationconsists of double-stranded RNA contaminant molecules.
 8. The purifiedRNA preparation of claim 1, further comprising a transfection reagent.9. The purified RNA preparation of claim 1, further comprising amammalian somatic cell, wherein at least some of said invitro-synthesized single-stranded mRNA molecules are inside said cell.10. The purified RNA preparation of claim 3, further comprising amammalian somatic cell, wherein at least some of said invitro-synthesized single-stranded mRNA molecules are inside said cell.11. The purified RNA preparation of claim 7, further comprising amammalian somatic cell, wherein at least some of said invitro-synthesized single-stranded mRNA molecules are inside said cell.12. A purified RNA preparation comprising a plurality of invitro-synthesized single-stranded mRNA molecules that encode 2 or more,3 or more, 4 or more, 5 or more, or 6 iPSC induction factors selectedfrom the group consisting of OCT4, SOX2, KLF4, LIN28, NANOG, and a MYCprotein selected from c-MYC, L-MYC and N-MYC; wherein said invitro-synthesized mRNA molecules: a) comprise a modified nucleotidecomprising a modified nucleoside in place of uridine that is selectedfrom the group consisting of pseudouridine that is not further modified(Ψ), 1-methyl-pseudouridine (m¹Ψ), 5-methyluridine (m⁵U),5-methoxyuridine (mo⁵U) and 2-thiouridine (s²U); and b) have beenpurified using a purification process that removes RNA contaminantmolecules that are immunogenic and toxic to a mammalian cell by inducingan innate immune response, such that said purified in vitro-synthesizedmRNA lacks immunogenicity, as can be determined using an in vitromonocyte-derived dendritic cell (MDDC) assay by measuring an amount ofIFN-α or TNF-α secreted by MDDCs transfected with said purified invitro-synthesized mRNA molecules that is no greater than the mean+thestandard error of the mean (SEM) amount of IFN-α or TNF-α secreted byMDDCs transfected with negative controls that do not contain RNA. 13.The purified RNA preparation of claim 12, wherein said invitro-synthesized mRNA molecules encode the iPSC induction factors: A)OCT4, SOX2, KLF4 and MYC; or B) OCT4, SOX2, KLF4, MYC and LIN28; or C)OCT4, SOX2, KLF4, MYC and NANOG; or D) OCT4, SOX2, KLF4, MYC, LIN28 andNANOG; wherein said MYC in A) D) is selected from c-MYC, L-MYC andN-MYC.
 14. The purified RNA preparation of claim 12, wherein saidpurification comprises at least one purification process selected fromthe group consisting of: A) HPLC or gravity flow column chromatography;B) treating a composition comprising said in vitro-synthesized RNAmolecules with one or more enzymes that specifically digest one or moreRNA contaminant molecules or contaminant DNA molecules; and C) treatinga composition comprising said in vitro-synthesized RNA molecules with aribonuclease III (RNase III) enzyme such that short RNase III digestionproducts are generated, and purifying said short RNase III digestionproducts away from said mRNA molecules, wherein, said purificationcomprises collecting and analyzing fractions from said purificationprocesses (i) for RNA contaminant molecules, (ii) for DNA contaminantmolecules, and (iii) for reduced immunogenicity using an in vitromonocyte-derived dendritic cell (MDDC) immunogenicity assay by measuringless secretion of IFN-α or TNF-α from MDDCs transfected with invitro-synthesized RNA molecules in more purified fractions.
 15. Thepurified RNA preparation of claim 12, wherein at least one of thefollowing applies: (i) greater than 98% of said mRNA molecules comprisea 5′ cap; (ii) said mRNA molecules comprise a cap with a cap0 structure;(iii) said mRNA molecules comprise a cap with a cap1 structure; (iv) allor substantially all said mRNA molecules comprise a poly(A) tail having50-200 or greater than 150 nucleotides; (v) said mRNA molecules compriseat least one particular sequence selected from the group consisting of:a heterologous 5′ UTR sequence, a Kozak sequence, an IRES sequence, anda 3′ UTR sequence; (vi) said 5′ UTR sequence or 3′ UTR sequence is froma Xenopus or human alpha globin or beta globin mRNA, or wherein said 5′UTR sequence is a sequence from a tobacco etch virus (TEV) RNA; (vii)said RNA contaminant molecules that are removed from said invitro-synthesized mRNA molecules using said purification process areselected from the group consisting of double-stranded RNA (dsRNA)molecules and un-capped mRNA molecules; (viii) less than 0.01% of thetotal RNA in said purified RNA preparation consists of double-strandedRNA contaminant molecules; (ix) said in vitro-synthesized mRNA moleculesfurther comprise m⁵C (5-methylcytidine) in place of cytidine; or (x)said composition further comprises a transfection reagent.
 16. Thepurified RNA preparation of claim 12, further comprising a mammaliansomatic cell, wherein at least some of said in vitro-synthesizedsingle-stranded mRNA molecules are inside said cell.
 17. The purifiedRNA preparation of claim 13, further comprising a mammalian somaticcell, wherein at least some of said in vitro-synthesized single-strandedmRNA molecules are inside said cell.
 18. A composition comprising amammalian somatic cell containing in vitro-synthesized mRNA molecules,wherein said in vitro-synthesized mRNA molecules encode the iPSCinduction factors: A) OCT4, SOX2, KLF4 and MYC, or B) OCT4, SOX2, KLF4,MYC and LIN28, or C) OCT4, SOX2, KLF4, MYC and NANOG, or D) OCT4, SOX2,KLF4, MYC, LIN28 and NANOG, wherein said MYC in A)-D) is selected fromc-MYC, L-MYC and N-MYC; and wherein said in-vitro synthesized mRNAmolecules comprise: (i) guanosine, (ii) adenosine, (iii) cytidine, and(iv) pseudouridine in place of uridine.
 19. The composition of claim 18,further comprising a transfection reagent.
 20. The composition of claim19, wherein the transfection agent is selected from a liposome, amicelle, a nanoparticle, a nanotube, and a cationic compound.